KR20080071615A - Botulinum nanoemulsions - Google Patents

Abstract

The embodiment described herein are related nanoemulsions comprising botulinum toxins. In one embodiment, the nanoemulsions are prepared by high pressure microfluidization and comprise a particle size distribution exclusively between 10 and 300 nm. The nanoemulsions contemplated by the present invention are useful for the cosmetic and medical treatment of muscular contracture states. For example, botulinum toxin may relax facial muscles such that skin wrinkles become smoother and less noticeable. Further, the present invention contemplates a cosmetic formulation that may be self-administered, for example, in the privacy of one's home and without medical supervision.

Description

Botulinum Nanoemulsion {BOTULINUM NANOEMULSIONS}

This application is related to USSN 60 / 741,139 (the '139 application), filed Dec. 1, 2005, which is the basis for claiming priority under the' 139 application under 35 USC 119 (e). The entire contents are incorporated herein by reference.

, 다한증 및 특정의 신경근육 질환을 치료하는 데 사용되어 오고 있다. 전형적으로, 보툴리눔 독소는 해당 부위 내로(즉, 주름 또는 띠 형성에 관여하는 관련 근육군 내로; 땀샘 함유 피부 내로, 기타 내로) 주사함으로써 전달된다.Botulinum toxin is used in cosmetic dermatology to treat various skin symptoms and diseases. For example, botulinum toxins may include wrinkles (e.g., hyperkinetic facial lines), broad plasma bands, decollete bands.

It has been used to treat hyperhidrosis, and certain neuromuscular disorders. Typically, botulinum toxin is delivered by injection into the site (ie, into the muscle groups involved in wrinkling or banding; into sweat glands containing skin, or into other).

Unfortunately, current botulinum toxin delivery methods cause a number of side effects. For example, improper injection techniques can damage tissue and / or deliver botulinum toxin to an unintended and / or unwanted location. In the area around the eyes, lid and brow ptosis are important side effects. Pain, hematoma, ecchymosis and bruising may also occur.

Techniques have been developed that can minimize certain side effects (e.g., techniques to cool the skin prior to injection to reduce pain, hematoma, ecchymosis and bruises), but improved systems and / or to deliver botulinum toxin There is still room for development of the formulation.

Summary of the Invention

The present invention provides nanoparticle compositions (eg nanoemulsions) containing botulinum toxin. This composition is useful, for example, in a variety of cosmetic and medical uses. In some embodiments of the invention, the botulinum nanoparticle composition is used to smooth out wrinkles. In some embodiments of the invention, the botulinum nanoparticle composition is used to treat hyperhidrosis. In some embodiments of the invention, the botulinum nanoparticle composition is used to treat muscle building and / or hyperactivity. Other uses of the botulinum nanoparticle compositions of the present invention are described herein and / or will be apparent to those skilled in the art.

In some embodiments of the invention, the botulinum nanoparticle composition is prepared with high shear force exposure; In some embodiments, the botulinum nanoparticle composition is prepared by microfluidization; In some embodiments, the botulinum nanoparticle composition is prepared by high pressure homogenization.

The botulinum nanoparticle compositions of the present invention may be administered by any available means, including but not limited to percutaneous means and injections (eg, intravenous, subcutaneous or intramuscular injection). The present invention includes the discovery that certain botulinum toxin nanoparticle compositions can be delivered transdermally without alteration or alteration of skin structure. For example, there is no need for abrasives or agents that erode or exacerbate the skin surface layer to achieve transdermal delivery of the botulinum toxin according to the present invention. In sum, in many embodiments, transdermal delivery of botulinum toxin is achieved without significant irritation to the skin.

In accordance with the present invention, percutaneous delivery can occur in any of a variety of forms. In some embodiments, the botulinum nanoparticle composition of the present invention is incorporated into a cream such that the cream is applied to the skin such that the botulinum toxin is administered to the subject. In some embodiments, the botulinum nanoparticle composition of the present invention is incorporated into a transdermal patch such that botulinum toxin is administered to the subject from the patch,

In some embodiments, the botulinum nanoparticle composition of the present invention has a difference between maximum and minimum diameters of about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70 Emulsions containing a population of particles of maximum and minimum diameters of no greater than 60 nm, no greater than 50 nm or less.

In some embodiments, the particles in the botulinum nanoparticle compositions of the present invention (eg, botulinum toxin containing particles) have a diameter of about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 130, 120 Less than 115, 110, 100, 90, 80 nm or less.

In some embodiments, the particles in the botulinum nanoparticle compositions of the invention (eg, botulinum toxin containing particles) are in the range of about 10 to about 600 nanometers in diameter. In some embodiments, the particles in the botulinum nanoparticle composition of the present invention have a diameter of about 10-300, 10-200, 10-150, 10-130, 10-120, 10-115, 10-110, 10-100 Or in the range of 10-90 nm.

In some embodiments, particles in a botulinum nanoparticle composition of the invention (eg, botulinum toxin containing particles) have an average particle diameter of about 300, 250, 200, 150, 130, 120, or 115, 110, 100, or 90 nm. Is below. In some embodiments, the average particle diameter is in the range of about 10-300, 50-250, 60-200, 65-150, 70-130 nm. In some embodiments, the average particle diameter is about 80-110 nm. In some embodiments, the average particle diameter is about 90-100 nm.

In some embodiments, the majority of particles in a composition of the present invention (eg, botulinum toxin containing particles) are smaller than or within the range of the sizes given. In some embodiments, the majority have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% particles in the composition. , 99.6%, 99.7%, 99.8%, 99.9% or more.

In some embodiments, nanoparticle compositions of the present invention are substantially free of particles (eg, botulinum toxin containing particles) that are substantially larger than about 120 nanometers in diameter. In some embodiments, the particles in the botulinum nanoparticle compositions of the invention (eg, botulinum toxin containing particles) are in the range of about 30 to about 115 nanometers in diameter. In some embodiments, most of the particles in the composition (eg, botulinum toxin containing particles) are within this range in diameter; In some embodiments, such compositions are substantially free of particles (eg, botulinum toxin containing particles) that are substantially larger than about 115 nanometers in diameter. In some embodiments, the particles in the botulinum nanoparticle compositions of the invention (eg, botulinum toxin containing particles) are in the range of about 30 to about 70 nanometers in diameter. In some embodiments, most of the particles in such compositions (eg, botulinum toxin containing particles) have a diameter within this range; In some embodiments, the composition is substantially free of particles larger than about 70 nanometers in diameter.

In some embodiments, nanoparticle compositions of the present invention have at least two different particle groups. For example, in some such embodiments, most of the particles in the nanoparticle compositions of the present invention are in the range of about 30-70 nm in diameter, while the second group of particles is in the range of 70-120 nm in diameter. In some such embodiments, the composition is not contaminated with particles larger than 120 nm in diameter.

In some embodiments, the botulinum toxin is partially or wholly present in the nanoparticles in the botulinum nanoparticle composition of the present invention; In some embodiments, the botulinum toxin is adsorbed onto the nanoparticle surface in the botulinum composition of the present invention; In some embodiments, the botulinum toxin is associated with the interface between the nanoparticles and the dispersion medium. In some embodiments, botulinum toxin is found at two or more of these positions in the nanoparticle composition.

In some embodiments of the invention, the botulinum toxin is selected from the group consisting of Form A, Form B, Form C _1, Form C _2, Form D, Form F and Form G. In some embodiments, the botulinum toxin is present as an isolated protein; In some embodiments, the botulinum toxin is present as part of a protein complex.

This application cites various patent publications, all of which are incorporated herein by reference.

Justice

Wear : As used herein, the term “wear” means any means of altering, rupturing, removing or destroying the upper layer of skin. In some embodiments, abrasion refers to a mechanical means of altering, rupturing, removing or destroying the upper skin layer. In some embodiments, abrasion refers to chemical means that alter, rupture, remove or destroy the upper layer of skin. In some instances, agents such as release agents, particulates (such as magnesium or aluminum particles), acids (such as alpha-hydroxy acids or beta-hydroxy acids), alcohols, and the like can cause wear. Generally, for example, Donovan (e.g., US Publication Nos. 2004/009180 and 2005/175636, and PCT Publication No. WO 04/06954), and Graham (e.g., US Pat. Nos. 6,939,852 and US Publications). 2006/093624) is expected to cause abrasion enhancers, such as described. Of course, those skilled in the art will appreciate that a particular agent may be abrasion when present in one concentration, or in combination with one or more other agents, but in different circumstances. Thus, whether or not a particular substance is an "abrasive" depends on the context. Abrasion can be readily assessed by those skilled in the art, for example, by monitoring for redness or irritation of the skin and / or histological examination of the skin showing alteration, rupture, removal or erosion of the stratum corneum.

Administration : As used herein, the term "administration" refers to the delivery of a nanoparticle composition of the present invention to a subject, and is not limited to any particular route, but rather refers to any route that the medical community accepts as appropriate. For example, the present invention is directed to a route of delivery or administration, including but not limited to transdermal, intramuscular or subcutaneous. In certain embodiments of the invention, administration is transdermal.

Biologically Active Agents : As used herein, the phrase “biologically active agent” means any substance that has activity in a living body and / or organism. For example, a substance that has a biological effect on the organism when administered to the organism is considered biologically active. In certain embodiments, when a protein or polypeptide is biologically active, a protein or polypeptide portion that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion. Botulinum toxin is a biologically active agent according to the present invention.

Botulinum nanoparticle composition : As used herein, the term “botulinum nanoparticle composition” means any nanoparticle composition wherein at least one nanoparticle comprises a botulinum toxin. Botulinum toxin may be present in the nanoparticles, on the nanoparticle surface, and / or in the micelle membrane defining the nanoparticles.

Botulinum toxin : As used herein, the term “botulinum toxin” refers to any neurotoxin produced by Clostridium botulinum . Unless stated otherwise, the term encompasses fragments or portions (eg light and / or heavy chains) of said neurotoxin that possess appropriate activity (eg muscle relaxation activity). As used herein, the phrase "botulinum toxin" includes botulinum toxin serotypes A, B, C, D, E, F and G. As used herein, botulinum toxin can also be purified as a botulinum toxin complex (ie, 300, 600, and 900 kD complexes) as well as a purified (ie, isolated) botulinum toxin (ie, about 150 kD). A "purified botulinum toxin" is defined as a botulinum toxin isolated or substantially isolated from other proteins, including proteins for the botulinum toxin complex. The purified toxin may be at least 95% pure, preferably at least 99% pure. Those skilled in the art will appreciate that the present invention is not limited to any particular source of botulinum toxin. For example, botulinum toxin for use in accordance with the present invention may be isolated from Clostridium botulinum, chemically synthesized, recombinantly produced (ie, host cells or organisms other than Clostridium botulinum). ), And otherwise.

Medicinal Cosmetics : As used herein, the term "cosmeceutical" refers to any medicament having both cosmetic and pharmaceutical properties (eg, benzoyl peroxide or retinol). Medicinal cosmetics are generally useful for external applications to improve complexion or overall appearance. The medicinal cosmetics may be applied as a composition which is exemplified but not limited to creams, oils, foams, sprays, liquids and the like. In some instances, carotenoids, phenolic compounds and / or water soluble antioxidants can act as medicinal cosmetics.

Cosmetic pharmaceutical preparations: means a composition that is topically applied which contains one or more medicaments with the term "cosmetic pharmaceutical preparations" is used herein cosmetic property. In some instances, cosmetic preparations may include emollients, nourishing lotion type emulsions, cleansing lotions, cleansing creams, skin milks, emollient lotions, massage creams, emollient creams, makeup bases, lipsticks, facial packs or facial gels, cleaning agents, e.g. For example shampoos, rinses, body cleansers, hair tonics or soaps, and / or dermatological compositions such as lotions, ointments, gels, creams, patches and / or sprays.

Cream : As used herein, the term “cream” refers to a composition that may be formulated typically for skin applications. Creams typically contain a matrix based on oils and / or fatty acids. Creams formulated according to the present invention may contain nanoparticles and (for example these nanoparticles) may be substantially completely penetrated through the skin during transdermal administration. Such creams may also serve as carriers for incorporating substances (eg botulinum toxin).

Dispersion Medium : As used herein, the term “dispersion medium” means a liquid medium in which particles (eg, nanoparticles) are dispersed. Generally, dispersions are formed when at least two immiscible substances are combined. "Oil-in-oil" dispersions are those in which oily particles are dispersed in an aqueous dispersion medium. A "water-in-oil" dispersion is a dispersion of aqueous particles in an oily dispersion medium. Those skilled in the art will appreciate that dispersions can be formed from any two immiscible media and will not be limited to strictly limited to the combination of aqueous and oily media. Thus, the term "dispersion medium" generally applies to any dispersion medium, although it is conventionally referred to in the "aqueous" and "oily" categories.

Encapsulation : As used herein, the term “encapsulation” (also “encapsulates” or “encapsulates”) is used to mean that the entity to be encapsulated is completely surrounded by another substance. In some instances, biologically active agents (eg, botulinum toxin) may be encapsulated within the nanoparticles in the emulsion of the invention. Such encapsulation can take place, for example, during the formation of nanoparticle compositions (eg nanoemulsions), for example microfluidization.

With : The phrase "with" as used herein means the co-delivery of two or more substances. In particular, according to the present invention, the phrase refers to the delivery of a biologically active agent with the nanoparticles and / or nanoparticle compositions of the present invention. The substance or medicament is such that the substance or medicament is combined with the nanoparticles and / or nanoparticle compositions; Encapsulated or completely surrounded by nanoparticles; Associated with the nanoparticle interface; And / or when adsorbed to the outer surface of the nanoparticles, are delivered with the nanoparticles. The substance or agent to be delivered with the nanoparticles and / or nanoparticle compositions may or may not be covalently bound to the nanoparticles and / or nanoparticle compositions. The substance or medicament to be delivered with the nanoparticle and / or nanoparticle composition may or may not be attached to the nanoparticle and / or nanoparticle composition by adsorptive force. In many embodiments of the invention, the biologically active agent delivered with the nanoparticles and / or nanoparticle compositions is botulinum toxin.

Microfluidization : The term “microfluidized” is generally used herein to refer to a composition that is exposed to high shear forces. In some embodiments of the invention, the composition is treated with equipment or devices known as "microfluidizers". However, in its broadest sense, the term microfluidization encompasses any composition that is exposed to high shear forces by any means. For example, high shear forces can be effected by cavitation or high pressure homogenization. Alternatively or additionally, the high shear force can be effected at high pressure, for example at an exposure of about 15,000 psi. In some embodiments, such high pressure is in the range of about 18,000 to about 26,000 psi; In some embodiments, it is in the range of about 20,000 to 25,000 psi. As shown, high shear forces can be implemented by, for example, passing through equipment such as a Microfluidizer ^® treatment device (Microfluidics Corporation / MFIC Corporation) or other similar device. The Microfluidizer ^® processing unit provides high pressure and hence high shear rates by accelerating the product at high speed through the microchannels to reduce the size to the nanoscale range. The fluid is separated in two and propagated through the microchannels at typical dimensions on the order of 75 microns at high speed (range 50-300 m / s). When the fluid exits the microchannel, it forms a jet that collides with the jet from the opposing microchannel. Fluid in the channel will experience high shear (10 ⁷ 1 / s or less), which is a higher order than conventional techniques. Jet conflict causes submicron level mixing to occur. Thus, high shear and impact are involved in particle size reduction and mixing of multiphase fluids in the Microfluidizer ^® technology. In some embodiments of the invention, the sample is “microfluidized” through high shear force exposure for a period of less than about 10 minutes. In some embodiments, the period of time is less than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute. In some embodiments, the period is about 1 to 2 minutes or less; In some embodiments, the duration is about 30 seconds. In some embodiments of the invention, the sample is “microfluidized” through a single exposure of high shear forces; This embodiment is referred to herein as a "single pass" microfluidization.

Nanoemulsions : Emulsions are customarily defined in the art as " systems composed of a liquid dispersed in an immiscible liquid with droplets larger than the colloid size, usually in the presence or absence of an emulsifier. Medline Plus Online Medical Dictionary, Merriam Webster (2005). The term "nanoemulsion" as used herein means an emulsion in which at least some droplets (or particles) have a diameter in the nanometer size range. As those skilled in the art will understand, nanoemulsions are characterized by droplets or particles that are thousands of times smaller than microemulsion droplets or particles.

Nanoparticles : As used herein, the term "nanoparticle" refers to any particle whose diameter is less than 1000 nanometers (nm). In some embodiments, the diameter of the nanoparticles is less than 300 nm as defined by the National Science Foundation. In some embodiments, the diameter of the nanoparticles is less than 100 nm as defined by the National Science Foundation. Those skilled in the art will appreciate that the term "nanoparticle" as used herein is used to refer to a dispersed phase in a dispersion or emulsion.

Nanoparticle Compositions : The term “nanoparticle composition” as used herein, means any composition comprising at least one nanoparticle. In some embodiments, nanoparticle compositions are a uniform collection of nanoparticles. Nanoparticle compositions disclosed herein are typically emulsions or dispersions. In some embodiments, nanoparticle compositions are stable. In some embodiments, nanoparticle compositions include one or more biologically active agents to be delivered with the nanoparticles. In some embodiments, nanoparticle compositions are nanoemulsions. Those skilled in the art will appreciate that nanoparticle compositions can be prepared according to any available means, including, for example, chemical or mechanical means. In some embodiments of the invention, nanoparticle compositions are prepared by microfluidizing a sample. In some embodiments of the invention, nanoparticle compositions are prepared without the use of toxic solvents and / or are substantially free of toxic solvents.

Not contaminated with : When the phrase "not contaminated with" is used herein with reference to a nanoparticle composition, it has the same meaning as "substantially free" and about 50% of the illustrated material. The nanoparticle composition contained below is shown. For example, if a nanoparticle composition is referred to as "substantially free" of particles outside of a specified range of diameters, less than about 50% of the particles having diameters outside of that range in the composition. In some embodiments, up to 25% of the particles are out of range. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% 4%, 3%, 2%, 1%, 0.5% or less of the particles have a diameter outside of the ranges indicated above.

Pharmaceutically Acceptable : As used herein, the term “pharmaceutically acceptable” refers to tissues of humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications, with reasonable benefit / hazardous ratios within normal medical judgment. Means a medicament suitable for use in contact with.

Premix : As used herein, the term "premix" refers to a combination of any of the ingredients used to subsequently produce the nanoparticle composition, or used according to the present invention. For example, a premix is a collection of optional components that produce nanoparticles according to the invention when applied to high shear forces. In some embodiments, the premix is a collection of components that produce nanoparticles, such as a uniform nanoparticle composition when applied at high shear. The premix often contains liquid dispersion medium and other components sufficient to produce nanoparticles in the dispersion medium. According to the invention, botulinum toxin may also be included in the premix. The premix may also include one or more surfactants and / or other agents. In some embodiments, the premix constitutes a solution. In some specific embodiments where the premix comprises botulinum toxin and / or other biologically active agent, the botulinum toxin (and / or other biologically active agent) is in solution before high shear force is applied to the premix.

Non-compliance : As used herein, the term “refractory” refers to any subject that does not respond with the expected clinical efficacy after delivery of a biologically active agent or pharmaceutical composition, as commonly observed by an active medical practitioner.

Self-administration : As used herein, the term "self-administration" refers to a situation in which a subject may administer the composition itself without the need for physician instructions. In some embodiments of the invention, self-administration may be performed outside of the clinical environment. By way of example, in some embodiments of the invention, the facial cosmetic cream may be administered by a subject at home in the subject.

Small molecule : In general, “small molecule” is understood in the art to be an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the small molecule is less than about 800 Daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D or 100 D. In some embodiments, the small molecule is a nonpolymer. In some embodiments, the small molecule is not a protein, peptide or amino acid. In some embodiments, the small molecule is not a nucleic acid or nucleotide. In some embodiments, the small molecule is not a saccharide or polysaccharide.

Stable : When the term “stable” is applied herein to a nanoparticle composition, it means that the composition retains one or more characteristics of its physical structure (eg, particle size range and / or distribution) for a period of time. . In some embodiments of the invention, the stable nanoparticle composition maintains for a period of time the average particle size, maximum particle size, particle size range and / or particle size distribution (i.e., the proportion of particles above and / or beyond the specified size range). will be. In some embodiments, the period of time is at least about 1 hour; In some embodiments, the duration is about 5 hours, 10 hours, one (1) day, one (1) week, two (2) weeks, one (1) months, two (2) months, three (3) months, It may be four (4) months, five (5) months, six (6) months, eight (8) months, ten (10) months, twelve (12) months, twenty-four (24) months or more. In some embodiments, the time period is in the range of about one (1) day to twenty-four (24) months, two (2) weeks to twelve (12) months, two (2) months to five (5) months, and the like. For example, if a group of nanoemulsion particles is subjected to long-term storage, temperature change, and / or pH change, and the majority of nanoparticles in the group maintain a diameter within a specified range (i.e., between about 10 and 120 nm), , Nanoemulsion is stable. In some of these groups, the majority are about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, More than 99.9% or more. In some embodiments of the invention, where the nanoparticle composition contains botulinum toxin and / or at least one other biologically active agent, the concentration of the biologically active agent (eg, botulinum toxin) is maintained over a specified period of time in the composition under specified conditions. If so, the nanoparticle composition is believed to be stable.

Subject : The term “subject” herein means any animal to which the nanoparticle composition of the invention can be delivered or administered. For example, the subject may be a human, dog, cat, cow, pig, horse, mouse, rat, gerbils rat, hamster and the like. In many embodiments of the invention, the subject is a human.

Symptoms are reduced : According to the present invention, when symptoms are reduced in size (eg intensity) or frequency for one or more symptoms of a particular disease, disorder or condition, “symptoms are reduced”. For clarity, delay in the onset of certain symptoms is considered a form of reducing the frequency of these symptoms. In some instances, when the condition is facial wrinkles, the symptoms of the condition are reduced when the depth and / or depth of one or more wrinkles in the selected area is reduced. If the condition is muscle building, the symptoms are reduced when the muscles are less tense and more relaxed. It is not intended that the present invention be limited only to cases where symptoms have been eliminated. The present invention specifically targets a treatment to cause one or more symptoms to be reduced (and thus "improved" in the subject's condition), although not completely eliminated.

A therapeutically effective amount : As used herein, the term “therapeutically effective amount” refers to an amount sufficient to treat the disease, disorder and / or condition when administered to a subject suffering from or susceptible to the disease, disorder and / or condition. it means. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount can be that amount that provides a particular pharmacological response of interest when administered or delivered to a significant number of subjects in need of treatment. In particular, it is to be understood that a particular subject may in fact be "non-compliant" to a "therapeutically effective amount". For example, non-compliance subjects may have low bioavailability such that clinical efficacy cannot be obtained. In some embodiments, the therapeutically effective amount can be an amount measured in one or more specific tissues.

Toxic Solvent : As used herein, the term "toxic solvent" refers to any substance that can alter, rupture, remove or destroy animal tissue. As will be appreciated by those skilled in the art, animal tissues may include live cells, dead cells, extracellular matrix, cell conjugates, biological molecules, and the like. To give some examples, toxic solvents include dimethyl sulfoxide, dimethyl acetimide, dimethyl formamide, chloroform, tetramethyl formamide, acetone, acetate and alkanes.

Treatment : As used herein, the term “treatment” (or “treat” or “treating”) refers in part to the manifestation of one or more symptoms or features of a particular disease, disorder and / or condition (eg, facial wrinkles) By any administration of a biologically active agent that completely alleviates, improves, revitalizes, inhibits, delays, reduces severity and / or reduces incidence. Such treatment may correspond to a subject that does not exhibit signs of an associated disease, disorder, and / or condition and / or a subject that exhibits only initial signs of a disease, disorder, and / or condition. Alternatively or additionally, such treatment may correspond to a subject exhibiting one or more established signs of related disease, disorder and / or condition.

Uniform : When the term “uniform” is used herein in connection with nanoparticle compositions, it means nanoparticle compositions in which the individual nanoparticles have a specific range of particle size sizes. For example, in some embodiments, the uniform nanoparticle composition has a difference between the minimum and maximum diameters of about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80 Not more than 70 nm, 60 nm, 50 nm or less. In some embodiments, the diameter of the particles (eg, botulinum toxin containing particles) in the uniform botulinum nanoparticle composition of the present invention is about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, Less than 130, 120, 115, 110, 100, 90, 80 nm or less. In some embodiments, the diameter of the particles (eg, botulinum toxin containing particles) in the uniform botulinum nanoparticle composition of the present invention is in the range of about 10 and about 600 nanometers. In some embodiments, the diameter of the particles in the uniform botulinum nanoparticle composition of the present invention is about 10-300, 10-200, 10-150, 10-130, 10-120, 10-115, 10-110, 10 In the range of -100 or 10-90 nm. In some embodiments, the average particle diameter of the particles (eg, botulinum toxin containing particles) in the botulinum nanoparticle composition of the present invention is about 300, 250, 200, 150, 130, 120, or 115, 110, 100, or 90 below nm. In some embodiments, the average particle diameter is in the range of about 10-300, 50-250, 60-200, 65-150, 70-130 nm. In some embodiments, the average particle diameter is about 80-110 nm. In some embodiments, the average particle diameter is about 90-100 nm. In some embodiments, the majority of particles (eg, botulinum toxin containing particles) in the uniform nanoparticle compositions of the present invention have a diameter below a certain size or within a certain range. In some embodiments, the majority have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% particles in the composition. , 99.6%, 99.7%, 99.8%, 99.9% or more. In some embodiments of the invention, the uniform nanoparticle composition consists of microfluidization of the sample.

Brief description of the drawings

Figure 1 shows an example of the particle size distribution of the microfluidized botulinum toxin nanoemulsion.

Figure 2 shows an example of the particle size distribution of the homogenized botulinum toxin microemulsion.

3 shows a patient attempting to lift the maximum eyebrow before topical administration (panel A) and after 2 weeks (panel B) of a composition of the present invention comprising a botulinum nanoparticle composition.

Description of Certain Preferred Embodiments

The present invention relates to botulinum toxin nanoemulsion compositions useful for cosmetic and medical treatments. Above all, the present invention provides a system for producing nanoparticle compositions containing botulinum toxin and methods of using such compositions in various situations. In one embodiment, the medical treatment relieves muscle building and / or overactivity; In another embodiment, the medical treatment relieves hyperhidrosis. In one embodiment, the cosmetic treatment straightens the skin. In one embodiment, the botulinum toxin nanoemulsion is prepared by microfluidization. Administration of the botulinum toxin nanoemulsion can be carried out by methods including but not limited to intramuscular injection or transdermal topical application.

Botulinum Toxin Biology

Botulinum Toxin (BTX) BTX is actually produced by Clostridium botulinum, an anaerobic Gram-positive bacterium, and is a powerful polypeptide neurotoxin. Among other things, BTX causes neuropathic disease called botulinum toxin in humans and animals. It is clear that BTX does not live through the intestinal lining and passes through it to attack peripheral motor neurons. Poisoning symptoms of botulinum toxin can lead to respiratory muscle paralysis from walking, swallowing and difficulty speaking, leading to death.

BTX-A is the most lethal natural biological agent known to humans. LD ₅₀ is about 50 picograms in female Swiss Webster mice (18-20 g) for commercialized BTX-A; This quantity is defined as 1 unit of BTX-A. On a molar basis, BTX-A is about 1.8 billion times more than diphtheria, about 600 million times than sodium cyanide, about 30 million times more than cobra poison and about 12 million times more lethal than cholera (Singh, et al , ed. , "Critical Aspects of Bacterial Protein Toxins" Natural toxins II , pp. 63-84, Plenum Press, New York, 1996).

Different serotypes of botulinum toxin differ in the severity and duration of the animal species they invade and the paralysis they cause. For example, BTX-A proved to be 500 times more potent than BTX-B when measured at the paralysis rate provided in rats. In addition, BTX-B has been demonstrated to be non-toxic to primates at a dose of 480 U / kg, about 12 times the primate LD ₅₀ for BTX-A. Moreover, botulinum toxin type B is known to have a shorter duration of activity and less potency than BTX- at the same dose level upon intramuscular injection.

Botulinum toxin apparently binds with high affinity to cholinergic motor neurons and migrates to neurons to block the release of acetylcholine.

Botulinum toxin is used in a clinical setting to treat certain neuromuscular disorders. In particular, BTX-A is approved by the US Food and Drug Administration for the treatment of blepharospasm, strabismus and semi-face spasms. Non-type A botulinum toxin serotypes clearly have a lower potency and / or shorter duration of activity compared to BTX-A. Clinical effects of peripheral intramuscular BTX-A are usually observed within 1 week of injection. The typical duration of a single intramuscular injection of BTX-A and relief of symptoms is on average about 3 months.

Although all botulinum toxin serotypes clearly inhibit the release of the neurotransmitter acetylcholine at neuromuscular junctions, they act as they affect different neurosecretory proteins and / or cleave these proteins at different sites. . For example, botulinum types A and E both cleave 25 kilodaltons (kD) of synaptosome-related protein (SNAP-25), but they target different amino acids in this protein. Botulinum toxin types B, D, F and G act on vesicle-associated proteins (VAMP, also called synaptobrebin), each serotype cleaves the protein at different sites. Finally, botulinum toxin C ₁ has been shown to cleave both syntaxin and SNAP-25. This difference in mechanism of action may affect the relative potency and / or duration of action of the various botulinum toxin serotypes. Significantly, the cytoplasm of islet B cells was at least SNAP-25 (Biochem J 1; 339 (pt 1): 159-65 (April 1999)) and synaptobrevin (Mov Disord 1995 May; 10 (3): 376).

The molecular weight of the botulinum toxin protein molecule for all seven known botulinum toxin serotypes is about 150 kD. Interestingly, botulinum toxin is released by Clostridial bacteria as a complex comprising a 150 kD botulinum toxin protein molecule with an associated non-toxin protein. In short, the BTX-A complex can be produced in 900 kD, 500 kD and 360 kD forms by spindle bacteria. Botulinum toxin types B and C ₁ are apparently produced only in the 500 kD complex. Botulinum toxin type D is produced in both 300 kD and 500 kD complexes. Finally, botulinum toxins E and F form only about 300 kD complexes.

BTX complexes (ie, compositions having a molecular weight greater than about 150 kD) are believed to contain non-toxic erythrocyte aggregate proteins and non-toxin and non-toxic non-erythrocyte aggregate proteins. These two non-toxin proteins (including the associated neurotoxin complex with the botulinum toxin molecule) can act to provide denaturation stability to the botulinum toxin molecule and protection against digestive acid when the toxin is ingested. It is also possible that larger (greater than about 150 kD molecular weight) botulinum toxin complexes can slow the diffusion rate of botulinum toxin away from the intramuscular injection site of the botulinum toxin complex.

BTX proteins or BTX complexes can be used according to the invention. Indeed, those skilled in the art will recognize that any portion or fragment of a BTX protein or complex that possesses the appropriate activity may be used as described herein.

In vitro investigations have shown that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brain stem tissue. In addition, botulinum toxin inhibits the induced release of glycine and glutamate in primary cultures of spinal cord neurons and inhibits the angular release of acetylcholine, dopamine, norepinephrine, CGRP and glutamate, of which the agent's botulinum toxin is a neurotransmitter in brain synaptosomes. Has been reported.

As discussed above, the botulinum toxin source is not critical to the present invention. However, for the sake of completeness, the inventors wish to disclose that a variety of sources, including commercial sources, are readily available for certain botulinum toxin formulations.

For example, a BTX or BTX complex can be obtained by establishing and growing a culture of Clostridium botulinum in a fermentor, followed by collecting and purifying the fermentation mixture according to known procedures. All botulinum toxin serotypes are first synthesized as inactive single chain proteins that are cleaved or nicked with proteases to become neuroactive. Thus, bacterial strains that allow botulinum toxin serotypes A and G to have endogenous proteases and serotypes A and G can be recovered primarily from the bacterial culture in their active form. In contrast, botulinum toxin serotypes C ₁ , D and E are synthesized by non-proteolytic strains and are therefore typically inert when recovered from culture. Serotypes B and F are synthesized by both proteolytic and nonproteolytic strains and thus can be harvested in both active or inactive forms. However, even proteolytic strains that provide, for example, BTX-A serotypes typically cleave only a portion of the toxin produced. The exact ratio of nicking molecules to non-nicking molecules may vary depending on incubation length and incubation temperature. Thus, for example, certain proportions of any agent of BTX-A toxin are likely to be inactive. The presence of inactive botulinum toxin molecules in the clinical formulation will contribute to the overall protein loading of the formulation and in some commercial botulinum toxin formulations will be involved in increasing antigenicity without contributing its clinical efficacy.

Higher crystalline botulinum toxin type A is generated from Hall A strain of Clostridium botulinum with different band patterns on ≥ 3 × 10 ⁷ U / mg, A ₂₆₀ / A ₂₇₈ of less than 0.60 and gel electrophoresis Can be. Known Shantz method can be used to obtain crystalline botulinum toxin including Form A (Shantz et al , 1992, Microbiol. Rev. , 56:80).

In general, botulinum toxin complexes can be isolated and purified in anaerobic culture by culturing Clostridium botulinum (eg, Form A) in an appropriate medium. Pure botulinum toxin upon isolation of the non-toxin protein, eg, purified botulinum toxin type A of about 150 kD molecular weight with a specific potency of 1-2 × 10 ⁸ LD ₅₀ U / mg or more; Purified botulinum toxin type B of about 156 kD molecular weight with a specific potency of at least 1-2 × 10 ⁸ LD ₅₀ U / mg; And known methods for obtaining purified botulinum toxin type F of about 155 kD molecular weight with a specific potency of 1-2 × 10 ⁷ LD ₅₀ U / mg or more.

Alternatively or additionally, prefabricated purified botulinum toxins and toxin complexes are described, for example, in List Biological Laboratories, Inc., Campbell, Calif .; the Center for Applied Microbiology and Research, Porton Down, UK; Wako ( Osaka, Japan) and Sigma Chemicals (St Louis, Mo.).

Pure botulinum toxin is unstable when administered in free solution and is generally not used to prepare pharmaceutical compositions. In addition, botulinum toxin complexes, such as toxin type A complexes, may be susceptible to denaturation due to surface denaturation, thermal and alkaline conditions. In some cases, the inactive toxin forms a toxoid protein, which may be immunogenic. The antibody produced allows the patient to be refractory to toxin injection.

In some embodiments, the present invention provides botulinum toxin nanoparticle compositions (eg, nanoemulsions) with improved stability of botulinum toxin as compared to the free solutions currently being administered. That is, in some embodiments, the botulinum toxin present in the nanoparticle compositions of the present invention is at least partially protected from at least one adverse condition such as heat, alkaline conditions, acidic conditions, degrading enzymes, host organism antibodies, and the like. Alternatively or additionally, the botulinum toxin present in the nanoparticle compositions of the present invention may exhibit less surface denaturation than comparable botulinum toxin formulations in free solution. As a specific example, 50 picograms of botulinum toxin in the microfluidized nanoemulsion according to the present invention will be protected from certain adverse conditions and the like that may result in surface denaturation.

Indeed, one aspect of the present invention includes the view that botulinum toxin can be stabilized by introduction into a nanoparticle composition. Those skilled in the art will appreciate that nanoparticle compositions according to one aspect of the present invention may be prepared by any available means.

The present invention also provides botulinum toxin nanoparticle compositions (eg, nanoemulsions) with improved skin penetration of botulinum toxin compared to the free solutions currently being administered. For example, the botulinum toxin introduced into the microfluidized nanoemulsion according to the present invention has improved membrane permeability compared to the free solution. In one embodiment, the minimum time between administration and intracellular accumulation will increase the efficacy of the method of administration and reduce side effects.

In addition, as demonstrated herein, the present invention provides botulinum toxin nanoparticle compositions in which botulinum toxin can penetrate the skin without the need to alter or destroy skin structure. For example, commercialization techniques for topical administration of biologically active agents typically require at least chemical, physical, electrical or other disruption of the outer skin layer. Such destruction can cause irritation, undesirable medical side effects and / or unwanted cosmetic consequences. The present invention provides botulinum toxin nanoparticle compositions that, when administered to the skin, allow botulinum toxin to penetrate the skin to provide a biological effect without significant or significantly irritating the skin and / or eroding the stratum corneum.

As with proteins, the biological activity of botulinum toxin (which is an intracellular peptidase) is generally affected by changes in three dimensional structures. In short, botulinum toxin type A can be detoxified by heat, various chemicals, surface stretching and surface drying. It has also been found that diluting toxin complexes obtained by known culture, fermentation and purification to much lower concentrations than toxins used in the formulation of pharmaceutical compositions can rapidly detoxify the toxins unless an appropriate stabilizer is present. . Dilution of toxins from milligram amounts to solutions containing nanograms per milliliter poses a significant challenge because, as such a significant dilution, certain toxins are rapidly lost. Since the toxin can be used for months or years after the toxin-containing pharmaceutical composition has been formulated, the liquid preparation of the toxin should be formulated with a stabilizer such as albumin.

As discussed above, the present invention provides a stabilized formulation of botulinum toxin. Despite the additional stability that can be imparted to the formulations of the present invention, in some embodiments of the present invention, the use of additional stabilizers is envisioned. For example, in some embodiments, at least one additional protein is used in conjunction with botulinum toxin. In some embodiments, such additional protein comprises albumin. In some embodiments, such additional proteins include one or more proteins naturally found in the botulinum toxin complex. Indeed, in some embodiments of the invention, a complete botulinum toxin complex is used. In some such embodiments, albumin is also used. Thus, in some embodiments, the present invention provides botulinum microfluidized nanoemulsions comprising albumin.

In some embodiments of the invention, the botulinum toxin used is BOTOX ^® . BOTOX ^® consists of purified botulinum toxin type A complex, albumin and sodium chloride, packaged in sterile vacuum dried form.

Botulinum toxin type A present in BOTOX ^® is prepared by culture of Clostridium botulinum Hall strain was grown in a medium containing NZ amine and yeast extract. The botulinum toxin type A complex is purified by a series of acid precipitations from the culture solution into a crystalline complex consisting of a high molecular weight active toxin protein and associated erythrocyte aggregate protein. The crystalline complex is redissolved in a solution containing saline and albumin and sterile filtered (0.2 micron) before vacuum drying. BOTOX ^® can be reconstituted with sterile unconserved saline prior to intramuscular injection. The BOTOX ^® for each vial will contain a Clostridium botulinum toxin type A purified neurotoxin complex, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride of about 100 units (U) as a sterile vacuum dried form without a preservative.

Currently, BOTOX ^® is usually reconstituted with 0.9% sodium chloride for administration by injection. Since BOTOX ^® can denature with bubbling or similar severe fluctuations, the diluent is slowly injected into the vial. It is recommended to administer BOTOX ^® as a free solution within 4 hours after reconstitution. It is also recommended to store the reconstituted BOTOX ^® in the refrigerator (ie, 2-8 ° C.) between reconstitution and injection. Reconstituted BOTOX ^® is transparent, colorless and has no specific substance.

BOTOX ^® has been reported for use in clinical settings as follows:

(1) BOTOX ^® from about 75 to 125 units per case for the treatment of cervical dystonia or more, intramuscular injection (multiple muscles);

(2) between the forehead wrinkle (forehead wrinkles) if the treatment, intramuscular state capitol BOTOX ^® 5 to 10 units (5 units injected intramuscularly and 10 units injected intramuscularly into each eyebrow frown wrinkles near the root);

(3), BOTOX ^® from about 30 to 80 unit case to treat constipation by intramuscular injection of articulation dudeong soon jageun window;

(4) about 1-5 units, which BOTOX ^® injections per muscle muscles When injected on the outer circumference near geompan whole eye and the outer circumference geompan all eyes near the lower lid of the upper lid treat blepharospasm;

5 for the Treatment of strabismus, is injected BOTOX ^® muscle of about 1-5 units in the outer eye muscles, jusaryang is dependent on the magnitude and a predetermined muscle paralysis be injected muscle (that is, the predetermined diopter correction amount).

(6) To treat arm stiffness after stroke by intramuscular injection of BOTOX ^® into five different flexion muscles:

(a) deep finger flexion muscle: 7.5 U to 30 U

(b) shallow finger flexion muscle: 7.5 U to 30 U

(c) posterior wrist flexion muscle: 10 U to 40 U

(d) Nose wrist flexors: 15 U to 60 U

(e) upper arm biceps: 50 U to 200 U.

Five of each muscle is indicated by the scan in the same therapeutic encounters and intramuscular injection of 90 U to 360 U BOTOX ^® of the arm near the bending muscle of the patients in each treatment meeting.

(7) To treat migraine headaches, BOTOX ^® injection of 25 U injected with cranial membranes (systemically into the tibialis, forehead and temporal muscles) causes migraine frequency, maximal severity, accompanying 3 months after 25 U injections. As measured by a decrease in the scale of vomiting and acute medication, this represents a significant advantage over vehicle as a prophylactic treatment of migraine headaches.

The present invention when the botulinum nanoparticle composition of the present invention containing the BOTOX ^® to be incorporated in the cream that is applied to the skin for transdermal delivery of the toxin, by injecting a botulinum toxin solution containing approximately equal to BOTOX ^® was observed histologically It has been demonstrated to produce a biological result comparable to that (ie wrinkle reduction).

The favorable clinical response of botulinum toxin type A has attracted interest in other botulinum toxin serotypes. Determine local muscle weakness efficacy, stability and antigenic potential for two commercially available botulinum type A preparations (BOTOX ^® and DYSPORT ^® ) and botulinum toxin B and F formulations (both available from Wako Chemicals (Japan)) An investigation was made to Botulinum toxin preparations were injected into the right calf muscle upper (0.5-200.0 units / kg) and muscle weakness was assessed using mouse toe abduction score (DAS) analysis. ED ₅₀ values were calculated from dose response curves.

Additional mice were injected intramuscularly for LD ₅₀ dose determination. The therapeutic index was calculated as LD ₅₀ / ED ₅₀ . BOTOX ^® (5.0 to 10.0 units / kg) or botulinum toxin type B (50.0 to 400.0 units / kg) were injected into the hind limbs of the different mouse groups and examined for muscle weakness and increased water consumption, the latter for dry mouth. It is a provisional model. Antigen potential was assessed by intramuscular injection in rabbits monthly (1.5 or 6.5 ng / kg for botulinum toxin type B or 0.15 ng / kg for BOTOX ^® ). Muscle weakness and duration peaks were dose related for all serotypes.

DAS ED ₅₀ values (units / kg) are as follows: BOTOX ^® : 6.7, DYSPORT ^® : 24.7, botulinum toxin type B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX ^® had a longer duration of action than botulinum toxin type B or botulinum toxin type F. The therapeutic index values are as follows: BOTOX ^® : 10.5, DYSPORT ^® : 6.3, botulinum toxin type B: 3.2. Water consumption is the botulinum toxin type B is even less effective in muscle weakness, BOTOX ^® is greater than in the case of injection of botulinum toxin type B is injected mice. Four months after infusion, antibodies to botulinum toxin type B were produced in two of four rabbits (treated at 1.5 ng / kg) and four of four rabbits (treated at 6.5 ng / kg). In a separate investigation, none of which have been demonstrated antibodies against botulinum toxin type A from the 9 BOTOX ^® treated rabbits. The DAS results suggest that the relative peak potency of botulinum toxin type A is equivalent to botulinum toxin type F and that botulinum toxin type F is greater than botulinum toxin type B. In the effective period, the botulinum toxin type A is longer than the botulinum toxin type B, and the effective period of the botulinum toxin type B is longer than the botulinum toxin type F. As can be seen from the therapeutic index value; Two commercially available formulations of botulinum toxin type A (BOTOX ^® and DYSPORT ^® ) are different. The increased water consumption observed after the injection of botulinum toxin type B into the hind limbs indicated that a clinically significant amount of this serotype entered the murine systemic circulation. The results also suggest that in order to achieve efficacy comparable to botulinum toxin type A, it is necessary to increase the dose of other serotypes investigated. However, an increase in capacity can impair stability. Furthermore, in rabbits, inde is antigenic than BOTOX ^® type B, which, perhaps, appear to be due to the high protein loading of injection to achieve effective dose of botulinum toxin Type B. Eur J Neurol 6 (Suppl 4): S3-S10 (1999).

As set forth herein, the present invention is directed to the use of botulinum toxin of any serotype. Those skilled in the art will readily be able to assess the suitability of a particular serotype for a particular use and, according to the techniques herein, the preparation of nanoparticle compositions containing such botulinum toxin. Accordingly, the present invention provides nanoparticle compositions containing botulinum toxin of any serotype, including compositions containing only botulinum toxin protein and compositions containing one or other proteins. In some embodiments, these other proteins include or consist of albumin; In some embodiments, botulinum toxin complex is used.

A source of a commercially available botulinum toxin which can be used according to the invention BOTOX ^®, DYSPORT ^® (human serum albumin and a Clostridium botulinum type A toxin hemagglutinin complex containing lactose; Ispen Limited, Berkshire UK) and / or MYOBLOC ^® and the like (botulinum toxin type B, primary consisting of human serum albumin, sodium succinate, and sodium chloride solution was used (pH 5.6), Elan Pharmaceuticals, Dublin, Ireland), but are not limited to these.

Nanoparticle Composition

As disclosed herein, the present invention provides compositions, in particular, nanoparticle compositions, including nanoparticle compositions containing botulinum toxin.

Generally, nanoparticle compositions are any composition comprising at least one nanoparticle. The botulinum nanoparticle composition is a nanoparticle composition containing botulinum toxin. Botulinum toxin is encapsulated or completely surrounded by one or more nanoparticles; Are compatible with the nanoparticle interface; It may be adsorbed on the outer surface of one or more nanoparticles. Botulinum toxin may or may not be covalently bound to the nanoparticles and / or nanoparticle compositions; Botulinum toxin may or may not be attached to nanoparticles and / or nanoparticle compositions by adsorptive forces.

In some embodiments, nanoparticle compositions of the invention are homogeneous aggregates of nanoparticles. For example, in some embodiments, the difference between the minimum and maximum diameters of the nanoparticles in the nanoparticle compositions of the present invention is about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 No more than 90, 80, 70, 60, 50 or less nm.

In some embodiments, the diameter of the particles (eg, botulinum toxin containing particles) in the botulinum nanoparticle composition of the present invention is about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 130, 120 Less than 115, 110, 100, 90, 80 nm or less.

In some embodiments, the diameter of the particles (eg, botulinum toxin containing particles) in the botulinum nanoparticle composition of the present invention is in the range of about 10 to about 600 nanometers. In some embodiments, the diameter of the particles in the botulinum nanoparticle composition of the present invention is about 10-300, 10-200, 10-150, 10-130, 10-120, 10-115, 10-110, 10-100 Or in the range of 10-90 nm.

In some embodiments, the average particle diameter of the particles (eg, botulinum toxin containing particles) in the botulinum nanoparticle composition of the present invention is about 300, 250, 200, 150, 130, 120, or 115, 110, 100, or 90 nm. Is below. In some embodiments, the average particle diameter is in the range of about 10-300, 50-250, 60-200, 65-150, 70-130 nm. In some embodiments, the average particle diameter is about 80-110 nm. In some embodiments, the average particle diameter is about 90-100 nm.

In some embodiments, the majority of particles in a composition of the present invention (eg, botulinum toxin containing particles) are smaller than or within the range of the sizes given. In some embodiments, the majority have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% particles in the composition. , 99.6%, 99.7%, 99.8%, 99.9% or more.

In some embodiments, nanoparticle compositions of the present invention have substantially no particles greater than 120 nm in diameter. In particular, in some embodiments, less than 50% of the nanoparticles in the nanoparticle compositions of the present invention have a diameter greater than 120 nm. In some embodiments, less than 25% of the particles have a diameter greater than 120 nm. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% 4%, 3%, 2%, 1%, 0.5% or less particles have a diameter in excess of 120 nm. In addition, in some embodiments, the nanoparticles in the nanoparticle compositions of the present invention are in the range of 10-120 nm in diameter.

Zeta potential is a measure of electrical potential at the shear plane. The shear plane separates a thin layer of liquid bound to a solid surface (e.g., the surface of the nanoparticles of the present invention) and shows an imaginary plane showing elastic behavior from the remaining liquid (e.g. liquid dispersion medium) exhibiting normal viscous behavior. to be. In some embodiments, nanoparticles of the invention have a zero potential in the range of -50 mV to +50 mV. In some embodiments, nanoparticles of the invention have a zero potential in the range of -25 mV to +25 mV. In some embodiments, nanoparticles of the invention have a zero potential in the range of −10 mV to +10 mV.

Nanoparticle compositions of the invention are typically emulsions or dispersions. In some embodiments, the composition is a “oil-in-water” dispersion (ie, a dispersion in which oily particles are dispersed in an aqueous dispersion medium); In some embodiments, the composition is a "water-in-oil" dispersion (ie, a dispersion in which aqueous particles are dispersed in an oily dispersion medium).

In some embodiments, nanoparticle compositions of the present invention do not require toxic solvents. In contrast, most conventional measures to induce the formation of nanoparticles in the composition use toxic (typically organic) solvents. In some embodiments, nanoparticle compositions of the present invention do not require a polymer. In contrast, most conventional measures for preparing compositions containing nanoparticle structures require polymers.

In some embodiments, nanoparticle compositions of the present invention have better tissue absorption and / or better biocompatibility than other nanoparticle compositions. For example, in some embodiments, the nanoparticle compositions of the present invention have better tissue absorbency than non-uniform nanoparticle compositions using one or more toxic (eg organic) solvents and / or using one or more polymers. Better or better biocompatibility.

In some embodiments, nanoparticle compositions of the invention (eg, botulinum nanoparticle compositions) are stable. In some embodiments of the invention, the stable nanoparticle composition maintains for a period of time the average particle size, maximum particle size, particle size range and / or particle size distribution (i.e., the proportion of particles above and / or beyond the specified size range). will be. In some embodiments, the period of time is at least about 1 hour; In some embodiments, the duration is about 5 hours, 10 hours, one (1) day, one (1) week, two (2) weeks, one (1) months, two (2) months, three (3) months, It may be four (4) months, five (5) months, six (6) months, eight (8) months, ten (10) months, twelve (12) months, twenty-four (24) months or more. In some embodiments, the time period is in the range of about one (1) day to twenty-four (24) months, two (2) weeks to twelve (12) months, two (2) months to five (5) months, and the like. For example, a group of nanoemulsion particles may be subject to long-term storage, temperature changes, and / or pH changes, and the majority of nanoparticles in the group maintain their diameters within a specified range (ie, between about 10 and 120 nm). , The nanoparticle composition is stable. In some of these groups, the majority are about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, More than 99.9% or more. In some embodiments of the invention, where the nanoparticle composition contains botulinum toxin and / or at least one other biologically active agent, the concentration of the biologically active agent (eg, botulinum toxin) is maintained over a specified period of time in the composition under specified conditions. If so, the nanoparticle composition is believed to be stable.

As described herein, nanoparticle compositions of the present invention are useful in a variety of medical, cosmetic and nutraceutical applications. Such compositions may be delivered to a subject by any available route, including but not limited to injection, oral delivery, transdermal delivery, and the like. In certain embodiments, the composition is delivered by injection. In certain embodiments, the composition is delivered transdermally.

It should be noted that the botulinum nanoparticle composition of the present invention is readily distinguishable from the other botulinum toxin containing compositions described. For example, Donovan has disclosed a formulation in which botulinum toxin is incorporated into lipid vesicles for percutaneous delivery (US Publication 2004/0009180). Such vesicles also require the introduction of enhancers such as alcohols to promote the absorption of botulinum toxin through the skin. Donovan also disclosed neurotoxins introduced into transferosomes, a modifying carrier containing lipids and membrane softeners (Hofer et al , 2000, World J. Sur g., 24: 1187; and US Pat. No. 6,165,500). Donovan specifically disclosed the preparation of sodium cholate liposomes impregnated with phosphatidyl choline + botulinum toxin.

Suvanprakorn et al. Also disclosed a suspension of liposome-encapsulated material in isolated macro-beads; In fact, the encapsulated one of hundreds of compounds can say that improved the "BOTOX ^®" (US Publication No. 2004/0224012 call). Methods for the preparation of these multilayer plate vesicle liposomes include lyophilization / rehydration and organic solution dehydration / aqueous rehydration. Conventional methods of preparing these liposomes are expected to provide microparticle sized vesicles.

Method of Preparation Nanoparticle Composition

In general, the nanoparticle compositions of the invention (eg, botulinum nanoparticle compositions) can be prepared by any available method. In some embodiments, nanoparticle compositions are prepared by chemical means. However, chemical means often require toxic (typically organic) solvents; In some embodiments, nanoparticle compositions are prepared according to the invention without the use of such solvents.

In certain embodiments of the invention, nanoparticle compositions are prepared by preparing a premix and applying the premix to high shear forces. The term "shear force" as used herein means a force parallel to the material plane as opposed to a force perpendicular to the material plane.

Any method in the art can be used to generate high shear forces. In some embodiments, cavitation is used to generate high shear forces. According to the present invention, the use of mechanical energy (ie high shear forces) can replace or minimize any requirement for using expensive and / or toxic chemical solvents; Increase the nanoparticle combination rate; It may increase the yield of nanoparticles generated in a particular ingredient mix and / or significantly reduce the total cost of preparing the nanoemulsion composition. In addition, in embodiments in which agents such as biologically active agents (eg, botulinum toxin) are introduced into the nanoparticle compositions of the present invention, the use of high shear forces may increase the loading capacity of the nanoparticles as compared to conventional methods of forming nanoparticles. Can be. In conventional methods, the loading of a medicament in or on the nanoparticles typically depends on the diffusion of the medicament into and / or onto the nanoparticles. The use of high shear forces in accordance with the present invention allows the production of smaller (eg on average) particles in the nanoparticle composition and / or narrower particle size distribution.

In some embodiments, the high shear force consists of continuous turbulence at high pressure exposure, such as high pressure, such as about 15,000 psi. In some embodiments, such high pressure is in the range of about 18,000 to about 26,000 psi; In some embodiments, it is in the range of about 20,000 to 25,000 psi.

In some embodiments, high shear force or high pressure may be applied by cavitation or high pressure homogenization.

In some embodiments, the high shear force can be effected by passing through equipment such as, for example, a Microfluidizer ^® treatment device (Microfluidics Corporation / MFIC Corporation) or other similar device. The Microfluidizer ^® processing unit provides high pressure and hence high shear rates by accelerating the product at high speed through the microchannels to reduce the size to the nanoscale range. The fluid is separated in two and propagated through the microchannels at typical dimensions on the order of 75 microns at high speed (range 50-300 m / s). When the fluid exits the microchannel, it forms a jet that collides with the jet from the opposing microchannel. Fluid in the channel will experience high shear (10 ⁷ 1 / s or less), which is a higher order than conventional techniques. Jet conflict causes submicron level mixing to occur. Thus, high shear and impact are involved in particle size reduction and mixing of multiphase fluids in the Microfluidizer ^® technology.

More generally, the microfluidizer can be any device that powers a single action booster pump. The booster pump amplifies to the adopted pressure, which in turn applies pressure to the output stream. As the pump moves through its pressure stroke, the product is driven at constant pressure through the interacting chamber. The product stream in the interacting chamber is accelerated at high speed to create high shear and impact forces so that a fixed product can produce a uniform nanoparticle composition (e.g. nanoemulsion) as the high speed product stream impinges on its own and abrasion resistant surfaces. Geometry microchannels are specially designed.

When the booster pump completes its pressure stroke, it reverses and attracts a new volume of product. At the end of the intake stroke, it inverts again and repeats the process by attracting the product at a constant pressure.

The product exiting the interacting chamber flows through a mounted heat exchanger that regulates the product to a predetermined temperature. At this time, the product can be recycled through the system for further processing or outward to the next step of the process (US Pat. Nos. 4,533,254; and 4,908,154).

In some embodiments of the invention, the sample is “microfluidized” through high shear force exposure for a period of less than about 10 minutes. In some embodiments, the period of time is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In some embodiments, the period is about 1 to 2 minutes or less; In some embodiments, the duration is about 30 seconds.

In some embodiments of the invention, the sample is “microfluidized” through a single exposure of high shear forces; This embodiment is referred to herein as a "single pass" microfluidization.

The present invention includes the view that application of the premix to high shear forces can produce nanoparticle compositions, and particularly uniform nanoparticle compositions.

In general, the premixes from which the nanoparticle compositions of the present invention are prepared by applying high shear forces are expected to contain at least two immiscible materials, one of which is a dispersion medium [ie, particles (eg nanoparticles) Liquid medium dispersed in the final nanoparticle composition. "Oil-in-water" dispersions are those in which oily particles are dispersed in an aqueous dispersion medium. A "water-in-oil" dispersion is one in which aqueous particles are dispersed in an oily dispersion medium. Those skilled in the art will appreciate that dispersions may be formed from any two immiscible media, and are not limited to strictly limited to the combination of aqueous and oily media. Thus, the term "dispersion medium" generally applies to any dispersion medium, although it is conventionally referred to in the "aqueous" and "oily" categories.

Thus, in some embodiments of the present invention, the premix will contain an aqueous dispersion medium and an oily medium that begins to disperse in nanoparticle form in the dispersion medium; In some embodiments of the present invention, the premix contains an aqueous medium that begins to disperse in the form of nanoparticles in the oily dispersion medium and the oily dispersion medium.

Those skilled in the art will be aware of suitable aqueous media that can be used as the dispersion medium or dispersion medium in accordance with the present invention. Typical examples of such aqueous media include, for example, water, saline (including phosphate buffered saline), water for injection, short chain alcohol, 5% dextrose, Ringer's solution (lactic acid-based Ringer's injection, lactic acid-added Ringer + 5%). Dextrose injections, acylated Ringer injections), Nomosol-M, Isolyte E and the like and combinations thereof.

Those skilled in the art will also know appropriate oily media that can be used as the dispersion medium or dispersion medium in accordance with the present invention. Representatives of such oily media include, for example, saturated and unsaturated almonds, apricot kernels, avocados, babassu palms, bergamots, black current seeds, borage, cades. , Chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flax seed , Geraniol, gourd, grape seed, hazelnut, hyssop, jojoba, cucui nut, labandine, lavender, lemon, litsea cubeba, mecademia nut , Mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin Seeds, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, poetry T (sea buckthorn), sesame seeds, shea butter (shea butter), silicone, soybean, sunflower, tea tree (tea tree), milk thistle, lettuce Baki (tsubaki), vetiver (vetiver), Walnut and Mac Ayu; Butyl stearate; Caprylic acid triglycerides; Capric acid triglycerides; Cyclomethicone; Diethyl sebacate; Dimethicone 360; Isopropyl myristate; Mineral oil; Octyldodecanol; Oleyl alcohol; Silicone oils; And combinations thereof.

In addition to the two immiscible media, the premixes according to the invention may comprise, for example, one or more biologically active agents (eg botulinum toxin) and / or one or more surfactants or emulsifiers. Suitable as such surfactants or emulsifiers are phosphoglycerides; Phosphatidylcholine; Dipalmitoyl phosphatidylcholine (DPPC); Dioleylphosphatidyl ethanolamine (DOPE); Dioleyloxypropyltriethylammonium (DOTMA); Dioleoylphosphatidylcholine; cholesterol; Cholesterol esters; Diacylglycerols; Diacylglycerol succinate; Diphosphatidyl glycerol (DPPG); Hexanedecanol; Fatty alcohols such as polyethylene glycol (PEG); Polyoxyethylene-9-lauryl ether; Surfactant fatty acids such as palmitic acid or oleic acid; fatty acid; Fatty acid monoglycerides; Fatty acid diglycerides; Fatty acid amides; Sorbitan trioleate (Span 85) glycocholate; Sorbitan monolaurate (Span 20); Polysorbate 20 (Tween-20); Polysorbate 60 (Tween-60); Polysorbate 65 (Tween-65); Polysorbate 80 (Tween-80); Polysorbate 85 (Tween-85); Polyoxyethylene monostearate; Supactin; Poloxomers; Sorbitan fatty acid esters such as sorbitan trioleate; lecithin; Lysocithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebroside; Dicetylphosphate; Dipalmitoylphosphatidylglycerol; Stearylamine; Dodecylamine; Hexadecylamine; Acetyl palmitate; Glycerol ricinoleate; Hexadecyl stearate; Isopropyl myristate; Tyloxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipids; Synthetic and / or natural detergents with high surfactant properties; Deoxycholate; Cyclodextrins; Chaotropic salts; Ion pairing agents; And combinations thereof, but is not limited thereto. The surfactant component may be a mixture of different surfactants. These surfactants can be extracted and purified from natural materials or can be prepared synthetically in the laboratory. In a preferred embodiment, the surfactant is commercially available.

In some embodiments of the invention, all components present in the final nanoparticle composition are present in the premix and are subjected to high shear forces to produce the nanoparticle composition. In some embodiments of the present invention, one or more of the components present in the final nanoparticle composition are excluded from the premix and / or present in smaller amounts in the premix than in the final nanoparticle composition. That is, in some embodiments of the present invention, after the premix is subjected to high shear forces, one or more materials are added to the nanoparticle composition.

In certain embodiments of the invention, the premix is prepared in solution prior to high shear force application. In particular, in the case of nanoparticle compositions comprising at least one biologically active agent (eg, botulinum toxin), it is often desirable to dissolve the biologically active agent in the premix before high shear force application. In short, in many embodiments, the biologically active agent is soluble in at least one medium (or combination of media used in the premix). In some embodiments of the invention, such dissolution requires heating; In other embodiments.

In some embodiments of the invention, the premix component may be assembled into particles prior to high shear force application. At least some of these particles may be microparticles or even nanoparticles. In some embodiments, nanoparticle compositions of the present invention are prepared from a premix, which is selected from the group comprising suspensions or microemulsions. However, in some embodiments, the particulated structure is not formed in the premix prior to high shear force application.

In certain embodiments of the invention, the relative amounts of the premix components are selected or adjusted to produce nanoparticles with certain properties. In some embodiments, the premix comprises oil and surfactant in a ratio ranging from 0.5 to 10. In some embodiments, the ratio of oil to surfactant is about 0.5: 1, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1. In some embodiments, the ratio of surfactant to oil is about 0.5: 1, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1. In some embodiments, the premix comprises oil and surfactant in a ratio ranging from 0.5 to 2. In some embodiments, the ratio of oil to surfactant is about 0.5: 1, 1: 1, or 2: 1. In some embodiments, the ratio of surfactant to oil is about 0.5: 1, 1: 1 or 2: 1. In certain embodiments, the ratio of oil to surfactant is about 1: 1.

In some embodiments, the proportion of oil in the premix ranges from 0% to 30%. In some embodiments, the proportion of oil in the premix is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 9%, 10%, 11%, 12%, 13%, 14 %, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% . In some embodiments, the proportion of oil is about 8%. In some embodiments the proportion of oil is about 5%.

In some embodiments, the proportion of surfactant in the premix ranges from 0% to 30%. In some embodiments, the proportion of surfactant in the premix is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 9%, 10%, 11%, 12%, 13%, 14 %, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% . In some embodiments the proportion of surfactant is about 8%. In some embodiments the proportion of surfactant is about 5%.

In some embodiments, nanoparticle compositions do not contain two or more oils. In some embodiments, nanoparticle compositions may comprise two or more oils. In some embodiments, nanoparticle compositions do not contain two or more surfactants. In some embodiments, nanoparticle compositions may comprise two or more surfactants.

In some embodiments, nanoparticle compositions consist essentially of water, oils, surfactants, and botulinum toxin. In some embodiments, nanoparticle compositions consist essentially of water, oils, surfactants, at least one botulinum toxin and at least one substance (eg, protein, salt, etc.) for producing and / or preserving nanoparticle compositions. do.

 In some embodiments, nanoparticle compositions consist of water, oils, surfactants, and botulinum toxin. In some embodiments, nanoparticle compositions are comprised of water, oils, surfactants, at least one botulinum toxin and at least one substance (eg, protein, salt, etc.) for producing and / or preserving nanoparticle compositions.

Method of Administration of Nanoparticle Composition

The present invention provides methods for delivering nanoparticle compositions (eg, botulinum nanoparticle compositions) for a variety of applications, including, for example, cosmetic, nutraceutical, and medical applications. Such nanoparticle compositions may comprise one or more biologically active agents. In many embodiments, nanoparticle compositions comprise botulinum toxin.

In some embodiments, the present invention is directed to a method of delivering a nanoparticle composition of the present invention, including but not limited to, transdermal, intramuscular or subcutaneous routes of administration. This route of administration is particularly advantageous for agents that seek local effects (eg, certain botulinum toxin nanoparticle compositions). However, there is no guarantee that the components in the formulation will subsequently be absorbed by the tissue.

In some embodiments of the invention, the formulations of the invention may be encapsulated, for example, using a carrier based on lipids, for example to facilitate entry into cells. However, the efficacy of a carrier based on lipids may include: i) lipid composition (ie, molecular size and charge); ii) the structure of any biologically active agent or other entity included in the composition (eg, molecular size and pH ionization); And iii) the general health of the subject. The present invention is directed to compositions and methods related to uniform microfluidized nanoemulsions that enhance the bioavailability of medicinal cosmetics (ie, botulinum toxin), including carriers based on two lipids.

The present invention specifically provides methods of administering botulinum toxin and in particular botulinum toxin nanoparticle compositions for the treatment of various disorders, diseases or conditions. The clinical effect of peripheral injection of botulinum toxin (ie intramuscular or subcutaneous) or topically applied transdermal administration usually occurs within one week. The typical symptom relief (ie, relaxation muscle paralysis) period from a single intramuscular injection of botulinum toxin type A may exist for up to 4 months or longer; The duration of clinical effect after percutaneous administration of the botulinum toxin according to the invention may be up to 4 months or longer, depending on the characteristics of the individual subject and / or the particular formulation of the botulinum nanoparticle formulation of the invention.

Those skilled in the art know that botulinum toxin is currently administered with almost no exception injections, especially liquid saline injections which are usually reconstituted from lyophilized preparations. As already discussed herein, botulinum toxin in the context of such formulations is particularly prone to destabilization and can result in loss of protein and / or loss of protein activity. Such instability is believed to be the result of protein denaturation, degradation, dimerization and / or polymerization. The most common agent known to have a botulinum stabilizing effect is human albumin. Possible immunological consequences of human-derived albumin have recently been reviewed (US Publication 2005/0238667). This publication suggests that recombinant albumin, saccharide based stabilizer and antioxidant amino acids can provide botulinum toxin with improved efficacy over natural albumin preparations.

As already discussed in herein, BOTOX ^® (sterile vacuum the purified Clostridium botulinum toxin type A complex, human serum albumin and sodium chloride packaged in dry form) is a preservative as of sterile saline (0.9% sodium chloride, injection grade) Without reconstitution for injection. Specifically, standard injection protocols involve drawing an appropriate amount of diluent into a syringe of the appropriate size. Since BOTOX ^® can be denatured by bubbling or similar severe fluctuations, the diluent is slowly injected into a vial containing a fixed amount of lyophilized OTOX ^® . Due to sterility, standard injection protocol involves administering within four hours after the reconfiguration the BOTOX ^® solution.

Problems with available botulinum toxin formulations (stability issues, sterility issues, etc.) are well known, but few improved formulations have been developed. Moreover, although invasive techniques are generally undesirable due to patient discomfort or the like, injections follow a standard approach to delivering botulinum toxin.

The present invention provides an improved botulinum toxin composition (eg, botulinum toxin nanoparticle composition) and further provides an improved method of delivering botulinum toxin. In particular, the present invention provides a method of delivering (by any available route) a botulinum nanoparticle composition and further provides a method of delivering botulinum toxin by a route other than injection.

In general, the botulinum nanoemulsion compositions of the present invention may be administered by any available means, including but not limited to parenteral, oral, transdermal, oral, ocular, intravaginal, rectal and the like. However, in certain embodiments, the composition comprises injection; In some embodiments, it is administered by subcutaneous injection, in some embodiments intramuscular injection, in some embodiments intravenous injection, and the like. In certain embodiments, the botulinum nanoparticle compositions of the invention are administered transdermally.

In certain embodiments, the present invention provides a method for transdermal administration of botulinum toxin. Human skin includes the dermis and epidermis. The epidermis has a number of tissue layers, ie, stratum corneum, clear layer, granule layer, play layer and basal layer (sizing inwardly from the outer surface of the skin).

The stratum corneum usually presents the most serious impediment to medication, and perhaps especially to the transdermal delivery of botulinum toxin. The stratum corneum is typically about 10-15 μm thick and consists of flat keratinocytes (keratocytes) arranged in several layers. The intercellular space between keratinocytes is full of lipid structures and may play an important role in infiltrating substances through the skin (Bauerova et al , 2001, European Journal of Drug Metabolism and Pharmacokinetics , 26:85).

The remaining epidermis below the stratum corneum is about 150 μm thick. The dermis is about 1 to 2 mm thick and is located under the epidermis. The dermis is stimulated by various capillary and neuronal processes.

Transdermal administration of a medicament is generally an investigational subject attempted to provide another route of administration of the medicament without the undesirable consequences associated with injection and oral delivery. For example, needles often cause local pain and there is the potential for exposed patients to blood infectious diseases. Oral administration often results in poor bioavailability of the drug because the patient's gastric environment is very acidic.

Efforts have been made to develop percutaneous administration techniques for certain drugs to address these drawbacks by providing noninvasive administration. In general, transdermal administration is preferred to reduce skin damage in patients. In sum, transdermal administration of drugs can reduce or eliminate pain associated with injections, reduce blood contamination, and improve the bioavailability of systemically introduced drugs.

Attempts for transdermal administration of conventional drugs have focused on increasing the permeability of the stratum corneum. Some attempts involve the use of chemical enhancers that increase the permeability of molecules through the skin. Some attempts involve the use of mechanical devices to bypass or remove stratum corneum portions. In addition, many attempts involve the use of ultrasound or iontophoresis to facilitate penetration of a medicament through the skin. In most cases, it is typically aimed at a drug, typically a small molecule, through the skin to allow the drug to pass through the intradermal capillary system, in which case the drug can be introduced systemically into the subject to achieve a therapeutic effect. .

Although small molecules are the main focus of transdermal administration techniques, it is important to note that large molecules such as polypeptides and protein complexes have also been shown to be transdermally administered. Erythropoietin, about 48 kD, has also been successfully transdermally administered with the aid of ultrasound (Mitragotri et al , 1995, Science , 269: 850; and US Pat. Nos. 5,814,599 and 6,002,961).

The present invention particularly provides a method for the transdermal administration of botulinum toxin, which does not require the use of abrasives or other destructive agents (whether chemical, mechanical, electrical, electromagnetic, etc.). To be precise, the inventors of the present invention have surprisingly found that the botulinum toxin introduced into the nanoparticle compositions of the present invention is effectively delivered transdermally without additional steps to penetrate or destroy the stratum corneum. The use of such agents or steps in conjunction with the botulinum nanoparticle compositions of the present invention is not necessarily excluded and not necessary in all embodiments of the present invention.

Accordingly, the present invention provides a method of administering a botulinum toxin by topically applying the botulinum nanoparticle composition of the present invention. In some embodiments, the botulinum nanoparticle compositions of the invention are applied directly to the skin to be absorbed through the epidermal layer. In some embodiments, the botulinum nanoparticle composition can penetrate the stratum corneum, the dermal pores and / or the upper skin layer, including the dermis, without the use of chemical or mechanical skin penetration enhancers or other agents that cause abrasion.

Those skilled in the art will appreciate that the compositions of the present invention for topical administration may contain cosmetic preparations such as emollients, nourishing lotion type emulsions, cleansing lotions, cleansing creams, skin milks, emollient lotions, massage creams, emollient creams, makeup bases, lipsticks, facial packs or It will be appreciated that they may be in the form of facial gels, cleaning agents such as shampoos, rinses, body cleansers, hair tonics or soaps, or dermatological compositions such as lotions, ointments, gels, creams, patches or sprays.

Compositions of the present invention for topical administration may be formulated and / or administered such that an amount of botulinum toxin from about 10 ⁻³ U / kg to 10 U / kg passes through the skin of the patient. In some embodiments, the composition is formulated and / or administered such that about 10 ⁻² U / kg to 1 U / kg percutaneously passes through the skin of the patient. In some embodiments, the composition is formulated and / or administered such that about 10 ⁻¹ U / kg to 1 U / kg passes through the patient's skin. In some embodiments, the composition is formulated and / or administered such that about 0.1 to about 5 units reach the subcutaneous destination through the skin of the patient.

Those skilled in the art will appreciate that units herein are biologically or bioactively equivalent to units defined by the commercial manufacturer of botulinum toxin.

The therapeutic effect of the botulinum toxin administered according to the present invention can last as long as there is an effect of the infusion solution. The effects of such infusion solutions can last up to about 4 months. In addition, long-term effects can be exerted for up to about 5 years by using synthetic polymer carriers that can hold botulinum toxins for slow release (US Pat. No. 6,312,708).

In one embodiment, the present invention provides topical preparations of botulinum toxin that can prevent potential complications including but not limited to systemic toxicity or botulinum toxin poisoning. In one embodiment, the dosage of botulinum toxin (including type A, B, C, D, E, F, or G) can range from a lower limit of about 1 unit to an upper limit of about 20,000 units, with minimal risk of side effects. have. The specific dosage may vary depending on the treatment symptoms and the treatment regimen used. For example, treatment of subcutaneous hyperactive muscle may require high transdermal doses of botulinum toxin (eg, 1000 to 20,000 units). In contrast, the treatment of nerve inflammation or hyperactive glands may require a relatively low transdermal dose of botulinum toxin (eg, about 1 unit to 1,000 units).

One embodiment of the invention is directed to a pharmaceutical composition containing a stabilized botulinum toxin for transdermal delivery to a human patient. Botulinum toxin is a group consisting of botulinum toxins A, B, C ₁ , D, E, F and G, isolated and / or purified (ie, about 150 kD) botulinum toxin, and botulinum toxin synthesized naturally or recombinantly Can be selected from. The composition may comprise about 1 to about 20,000 units of botulinum toxin, and the composition may comprise an amount of botulinum toxin sufficient to achieve a therapeutic effect that lasts from 1 month to 5 years.

In some embodiments, the present invention provides topical formulations of botulinum toxin (eg, of botulinum nanoparticle compositions) that allow botulinum toxin to penetrate through the subject's skin without significant infiltration through blood vessels. . For example, in some embodiments of the invention, upon application of the topical and / or transdermal formulations of the invention, less than about 25%, or even less than about 5%, of botulinum toxin present in the pharmaceutical composition is directed to the blood vessel. Infiltrate

Those skilled in the art will appreciate that a composition of the present invention that achieves percutaneous administration of botulinum toxin can be incorporated into a device, such as a patch.

Various transdermal patch structures are known in the art; Those skilled in the art will appreciate that the botulinum nanoparticle compositions of the present invention can be readily incorporated into any of these various structures. In some embodiments, the transdermal patch may further comprise a plurality of needles extending from one side of the patch applied to the skin, wherein the needles extend from the patch to protrude through the stratum corneum of the skin. In some embodiments, the needle does not disrupt blood vessels.

In some embodiments of the invention, a botulinum toxin (eg, botulinum nanoparticle composition) may be provided to the depot in the patch such that when pressure is applied to the patch, the botulinum toxin is directed from the patch (optionally through a needle) to the stratum corneum.

In some embodiments of the invention, the transdermal patch includes an adhesive. Some examples of adhesive patches are well known (see, eg, US Patent Des. 296,006; 6,010,715; 5,591,767; 5,008,110; 5,683,712; 5,948,433; and 5,965,154). Adhesive patches are generally characterized by having an adhesive layer to be applied to the skin of a person, a depot or reservoir for holding the medicament and an outer surface to prevent the medicament from leaking from the depot. The outer surface of the patch is typically non-adhesive.

According to the present invention, the neurotoxin is introduced into the patch so that the neurotoxin remains stable for a long time. For example, neurotoxins may be present in the botulinum nanoparticle compositions of the invention. Alternatively or additionally, the neurotoxins can be incorporated into a polymeric matrix that stabilizes the neurotoxins and diffuses the neurotoxins from the substrate and patches. Neurotoxins may also be introduced into the adhesive layer of the patch so that when the patch is applied to the skin, the neurotoxin can diffuse through the skin. In one embodiment, the adhesive layer can be thermally activated, in which case a temperature of about 37 ° C. slowly liquefies the adhesive so that the neurotoxin diffuses through the skin. The adhesive may be tacky when stored below 37 ° C., and once applied to the skin, the adhesive loses its tackiness as it liquefies. Administration of the toxin is complete when the patch no longer adheres to the skin.

Those skilled in the art will appreciate that transdermal patches are an example of a device in which the botulinum nanoparticle composition of the present invention may be administered. In some other instances, the device can be used to cause undesirable finger paralysis so that the composition can be applied without first applying the composition to a finger. Suitable devices include spatula, swabs, needleless syringes and adhesive patches. The use of a spatula or swab or the like may require a device to be inserted into a container containing the composition. The use of a syringe can be achieved by filling the syringe with a composition. The composition can then be topically spread by a spatula or swab or released from the syringe onto the human skin.

In many embodiments of the invention, it may be desirable to limit the delivery of botulinum toxin only to the intended delivery region. In some embodiments, such limited delivery can be achieved using the botulinum nanoparticle composition in an application device that allows the composition of the present invention to be applied to a target site on the skin without being applied to areas of non-target sites of the skin. Clearly, transdermal patches can be used for this purpose. Alternatively or additionally, if the botulinum toxin is applied locally only to the selected area, the other area may be covered, pretreated, or otherwise protected from exposure.

Therapeutic uses of botulinum toxin

As described herein, many embodiments of the present invention include delivering a botulinum toxin of the nanoparticle composition environment to a subject. Such delivery is particularly useful in a variety of environments, including certain cosmetic and medical applications. This particular application is described in more detail below.

Cosmetology  Usage

Botulinum toxin A (BTXA) is a widely used drug in cosmetic dermatology. The side effect observed with BTXA with respect to cosmetic use is that it has a significant impact on patient compliance. Currently, BTXA is administered in a clinical setting by medical personnel because BTXA requires trained agents to be administered by injection and the primary tool for preventing side effects caused by BTXA is knowledge and skill. Since most unwanted effects are accompanied by inaccurate techniques, the use of accurate injection techniques is essential. Human anatomy (eg facial and extra-facial muscle) knowledge is important to physicians in selecting the optimal dose, time and technique.

The most common side effects of the current procedure for administering BTXA are pain and hematoma. For example, when the BTXA solution is administered to the peri-eye area by injection, eyelid and brow sagging are common side effects. Side effects such as pain, hematoma, ecchymosis and bruises can also occur in the upper, lower and lower facial areas. Other possible side effects include, but are not limited to, headaches and possible interactions with the companion drug. In addition to using appropriate techniques of dilution, storage and injection, careful exclusion of patients with any contraindications has been proposed to prevent the most unwanted side effects. Pain, hematoma, ecchymosis and bruises can be prevented by cooling the skin before and after BTXA injection. Stomach eyelid sagging can be partially corrected by using apraclonidine or phenylephrine eye drops (Wollina et al , 2005, Am. J. Clin . Dermatol , 6: 141). However, serious side effects still exist in the present manner.

In contrast, the present invention provides methods and compositions for the safe and effective administration of botulinum toxin in a manner that minimizes adverse side effects. In one embodiment, the invention is directed to a method of administering botulinum locally and / or locally as a composition comprising a nanoparticle composition, such as, for example, microfluidized nanoemulsions. In one embodiment, the composition is formulated as a cream, ointment, oil, foam, spray or gel.

Those skilled in the art will appreciate that the botulinum nanoparticle compositions of the present invention may be formulated with any of a variety of cosmetically acceptable media in cosmetic preparations, such as liquids, creams, emulsions, gels, thickening lotions or powders; These include water and also any cosmetically acceptable solvent, in particular monoalcohols such as alkanols having 1 to 8 carbon atoms (ethanol, isopropanol, benzyl alcohol and phenylethyl alcohol, etc.), polyalcohols such as alkylene glycols (glycerine, Ethylene glycol and propylene glycol, etc.) and glycol ethers such as mono-, di- and tri-ethylene glycol monoalkyl ethers, such as ethylene glycol monomethyl ether and diethylene glycol monomethyl ether, may be contained alone or in combination. Can be. Such components may be present in large proportions, for example up to 70% by weight relative to the weight of the total composition.

The cosmetic preparations comprising the botulinum nanoparticle compositions of the present invention may comprise at least one filler which is particularly desirable for the individual to have smooth skin in order to obtain a matte product. The term "filler" is a solid at room temperature and atmospheric pressure, used alone or in combination, and does not chemically react with the various components of the composition, and these components apply at temperatures above room temperature, in particular their softening point or their melting point. Even if so, any particles that do not dissolve in these components. Such inert fillers typically have melting points above at least 170 ° C. and more preferably above 200 ° C.

Fillers may be adsorbent or nonadsorbent, ie they may also adsorb biological substances, in particular secreted by the oils and skin of the composition. In some embodiments, the filler is particulate and has an apparent diameter of 0.01 to 150 μm, preferably 0.5 to 120 μm, and more preferably 1 to 80 μm. The apparent diameter corresponds to the diameter of the circle in which the element particles are inscribed along their smallest dimension (thin plate thickness).

Wrinkle treatment

Facial wrinkles, including foreheads, foreheads, skin rhytids, and / or periorbital regions, are a common aesthetic problem and are believed to be associated with underlying facial muscle overactivity. For example, the occurrence of glabellar wrinkles is associated with the dynamics of at least some basic eyelids, eyebrow wrinkles, and eyelid muscles. Facial wrinkles are considered problematic because they show an aging appearance. In some cases, they may also be mistaken for expressions of negative emotions (eg anger, anxiety, sadness), fatigue or stress.

Recently, injections of botulinum toxin solutions have become one of the most popular treatment regimens for excessive facial wrinkles. After injection, toxins act to neutralize and weaken the facial muscles of the face. This apparently appears to reduce or eliminate the appearance of wrinkles. Sadick NS., "The cosmetic use of botulimun toxin type B in upper face" Clin Dermatol. 22 (l): 29-33 (2004).

The first cosmetic use of the botulinum toxin solution was to treat frown lines in the forehead (Carruthers et al , 1992, J. Dermatol. Surg Oncol , 18:17). In addition, the infusion of BTX solution into the wide neck was noticed by lifting the lips (Brandt et al , 1998, Dermatol. Surg ., 24: 1232). Injecting BTX solution at the lower jaw point is also done to treat prominent jaw wrinkles (Carruthers et al , "Cosmetic Uses of Botulimun A Exotoxin," pp. 325-48, Advances in Dermatology , James, et al , eds. , Mosby-Yearbook, Chicago, 1997).

The present invention provides nanoparticle compositions for treating facial wrinkles and / or unsightly complexion (such as due to overactivity of the underlying facial muscles). Of course, the principles and / or compositions related to the treatment of facial wrinkles and / or complexions may likewise be applied to undesirable lines or wrinkles due to muscle activity elsewhere in the body (eg, the neckline, etc.). In some embodiments, nanoparticle compositions of the invention for use in treating wrinkles comprise one or more neuroplegic toxins; In some embodiments, this toxin may block facial muscle activity; In some embodiments, the toxin comprises botulinum toxin (BTX). In some embodiments, the invention is directed to administering a microfluidized botulinum toxin nanoemulsion to facial wrinkles.

Recently, it has been proposed that repeated use of a botulinum type A toxin treatment may delay the occurrence of facial wrinkles and / or wrinkle lines by prolonged use (Binder, 2006, Arch. Facial Plast. Surg ., 8: 426). However, repeated injections are painful for the patient and carry the risk of injecting into unintended muscle groups and causing adverse side effects (eg sagging). In some embodiments, the botulinum nanoemulsion is applied to the face and / or neck for a long time to delay the occurrence of wrinkles or wrinkles on the face (or neck). In some embodiments, botulinum nanoemulsions are applied at regular intervals over the face and / or neck to delay the occurrence of wrinkles or wrinkles on the face. In some embodiments, botulinum toxin is applied at regular intervals over a period of more than six months to the face and / or neck to delay the occurrence of wrinkles or wrinkles on the face. In some embodiments, botulinum toxin is applied at regular intervals over a period of more than one year to the face and / or neck to delay the occurrence of wrinkles or wrinkles on the face. In some embodiments, botulinum toxin is applied at regular intervals over a period of more than 5 years to the face and / or neck to delay the occurrence of wrinkles or wrinkles on the face. In some embodiments, botulinum toxin is applied at regular intervals over a period of more than 10 years to the face and / or neck to delay the occurrence of wrinkles or wrinkles on the face.

Hyperkinetic facial wrinkles

Injection of botulinum toxin type B (BTX-B) was evaluated for the management of excessive facial wrinkles. For example, the dorsal, circumferential or forehead muscles of 24 patients were treated with 400-800 units of BTX-B. Wrinkle improvement score (WIS) and numerically rated Rated Numeric Kinetic Line Scale (RNKLS) can be used to assess facial wrinkle line improvement. There is an investigation that reported onset of effect started within 72 hours. WIS and RNKLS for all sites were statistically better after treatment and this effect lasted 8 weeks. In general, patients showed moderate improvement (grade 2) for WIS and two-point improvement in RNKLS (Ramirez et al , 2002, Otolaryngol. Head Neck Surg ., 126: 459).

In some embodiments, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to hyperkinetic facial wrinkles. In some embodiments, the invention is directed to administering a botulinum nanoparticle composition to a facial area where wrinkles typically occur prior to wrinkle formation. Repeated administration may eventually delay the development of wrinkles and / or reduce the strength or severity of wrinkles (Binder, 2006, Arch. Facila Plast. Surg ., 8: 426).

Wide neck strap

The wide neck is a wide, thin layer of muscle located on each side of the neck just below the thin fascia, which belongs to the facial muscle group, which develops the distribution of facial nerves, pulls the lower lip and corners sideways, and when moved strongly, the neck expands and its skin Pull upwards.

Injections of botulinum toxin Z have been reported to treat bloated wide neck strips (ie typically referred to as older necks) that slump down. Classification lines (I to IV) based on transverse neck wrinkles, wide neck bands and skin relaxation can categorize the degree of deformation and provide guidance as a suggested amount of botulinum. For example, type II means mild transverse neck wrinkles, light and thin wide root muscle relaxation, and mild skin relaxation, and type III means moderate transverse neck wrinkles, thick moderate wide neck root relaxation, and moderate skin relaxation. (Matarasso et al , 1999, Plast. Reconstr. Surg , 103: 645).

In one embodiment, the invention is directed to the administration of a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a wide neck strip.

Medical use

Neuromuscular diseases

BTX, produced by the Clostridium botulinum bacteria, reversibly paralyzes the striated muscle when administered at sub-digestive doses. BTX is a muscle spasm and / or buildup that includes various forms of paralysis, facial buildup, dystonia, semi-facial spasms, tremors, stiffness (such as due to multiple sclerosis), post-orbital muscles, and other various ophthalmic symptoms Has been used to treat a number of neuromuscular diseases and symptoms, including (Carruthers et al , 1996, J Am. Acad. Dermatol , 34: 788).

Facial paralysis

Injection of BTX into one muscle group of the patient's face has been reported to be used to treat facial cooperative movement and vertical asymmetry caused by facial nerve palsy (Armstrong et al , 1996, Clin. Otolaryngol ., 21:15). In the post-procedure, the mouth-related levator muscles, along with various eye-related muscles on the normal side of the patient's face, to influence the overall vertical asymmetry of the patient's face as a reward for the effect of nerve paralysis on the untreated face face, Large cheekbones, mouth tail pulls and mouth tail downs were all treated in one group.

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a rigid facial muscle.

Eyelid spasms

Blepharospasm is diagnosed as a repetitive and rhythmic contractile response of the eyelid muscles (ie also known as eyelid spasm). In some cases, it may be repeated that the eyelids are closed (or nearly closed) and that they float again. The cause of this condition can usually be caused by fatigue, stress and / or caffeine. However, once spasms start, they can continue irregularly for several days.

More severe contractions may occur that can completely close the eyelids. This worsened condition may be due to irritation of the eye surface (cornea) or the eyelid lining (conjunctiva). This type of eyelid single contraction lasts longer, often very uncomfortable, and can also cause the eyelids to close completely.

Symptoms of blepharospasm include, but are not limited to, repetitive and uncontrollable single contraction or spasm of the eyelids (usually the upper eyelids), photosensitive or blurred vision.

In some embodiments, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to the eyelid muscle.

Cerebral palsy

Cerebral palsy includes a group of disorders characterized by loss of motility or other loss of nerve function. This disorder is due to brain damage that occurs during fetal development or early delivery. Cerebral palsy can be attributed to damage to the cerebrum (the largest area of the brain involved in high intelligence, sensory and voluntary muscle activity).

Cerebral damage can lead to loss of nerve function in a wide variety of different areas. Typical findings of CP are stiffness (increased muscle tension) that can affect one limb, one side of the body (rigid half paralysis), both legs (rigid bilateral paralysis), or both arms and legs (rigid quadriplegia). In addition, there may be partial or complete loss of mobility (paralysis), paresthesia and hearing and visual defects. More than language is common and seizures can occur.

The intelligent functions of CP patients can range from extremely weak normals to severe mental retardation. Symptoms are usually evident before 2 years of age, and in severe cases, may occur even at the beginning of 3 months. Cerebral palsy is a non-progressive type of encephalopathy (brain damage), and the symptoms directly caused by the disease do not worsen.

Cerebral palsy classifications include stiffness, dyskinesia, incoordination and mixed form. Stiff cerebral palsy accounts for about 50% of cases. Movement disorders (disorderly movement) Cerebral palsy affects about 20%. This includes the occurrence of abnormal movements (torsional, turbulent or other motility). Coordination Cerebral palsy includes tremor, anxiety gait, loss of coordination, and abnormal exercise. This affects about 10%. The remaining 20% are classified as mixed, with any combination of the above symptoms.

Cerebral palsy symptoms include seizures, muscle contractions, difficulty in sucking or ingestion, irregular breathing, delayed motor function development, such as reaching, sitting, rolling, quadrupeds, walking, motor mental retardation, mental retardation, speech disorders (stuttering), Vision impairment, hearing impairment, stiffness, progressive joint build-up, range of motion limitation, or stereotypes may be, but are not limited to.

Botulinum toxin is effective in treating children with cerebral palsy and other hypermuscular strains by reducing deformation, promoting function, improving motor control, and stretching shortened muscles. In children with focal hypertension, botulinum toxin provides significant but temporary repeatable changes that affect rehabilitation. The investigation quickly captured the positive effects of toxins on disability and functional limitations. The long-term use of botulinum toxin and the role of toxins in the life of humans with pediatric hypertension disorders have already been demonstrated (Gaebler-Spira et al , 2003, Phys. Med. Rehabil. Clin. N. Am ., 14: 703 ).

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a patient exhibiting symptoms of hypertension. In one embodiment, the hypermuscular tension includes cerebral palsy. In one embodiment, the patient is a pediatric.

Strabismus

Strabismus includes the alignment of one eye relative to the other, and is also referred to as crossed eye, esotropy, exotropia, sapal or walleye. Lives are thought to be caused by lack of coordination between eyes. As a result, the eyes look in different directions and at the same time fail to focus on a single point.

Most pediatric strabismus is unknown. In more than half of these cases, problems occur shortly after birth (congenital strabismus). If both eyes fail to focus on the same image, the brain ignores input into one eye. If this continues, the eye the brain ignores will never be seen. This loss of vision is called amblyopia and is often associated with strabismus.

Acquired strabismus in adults can be attributed to orbital damage to the eye or brain, including obstructive head injury and stroke. People with diabetes frequently lose the circulatory system, leading to acquired paralytic strabismus. Loss of vision of one eye resulting from any cause will generally cause the eye to turn outward (exotropia). Since the adult brain is already visually developed, problems with amblyopia that ignore the brain input into one eye do not occur in adult strabismus.

Symptoms of strabismus include, but are not limited to, eyes that appear crossed, eyes that are not aligned in the same direction, uncooperative eye movement, double vision, or loss of cognition in one eye.

Long-term results of botulinum therapy have been reported in patients with acquired esotropia. 68 children with acquired esotropia (ages 8 to 64 months) were included in the prospective study. Botulinum toxin A was injected into both rectus muscles. Motor and sensory conditions 1 and 2 weeks after the final injection; 3, 6 and 12 months; And evaluated every year.

After an average of 4.8 years of follow-up from the last injection, exercise success was observed in 36 children (52.9%) with one injection, and 48 (70.6%) and 60 (88.2%) after 2 and 3 injections, respectively. Increased. 58 (70.6%) patients had at least peripheral fusion (category 1 of category 1) and 32 (47.1%) had stereoscopic vision of at least 400 seconds (category 2 of category 2). Highly primitive, milder amblyopia and incineration esotropia are the best predictors of motor success. Lowest amblyopia and favorable motor alignment resulted in better binocular results.

Botulinum toxin may be an effective long-term treatment for acquired esotropia. This is especially useful for children with high primitive, lowest amblyopia and small esotropia (Tejedor et al , 2001, Investigative Ophthalmology and Visual Science , 42: 2542).

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a patient with strabismic symptoms. In one embodiment, the patient is a pediatric.

Dystonia

Myotonic dysfunction is a medical condition involving a slow involuntary torsional movement. Uncontrolled or slow movement is defined as a muscular tension disorder (usually in large muscle groups) that causes slow involuntary contractions of the head, limbs, torso or neck (ie, cervical dystonia). Slow wave torsional movement of muscles (abnormal movements) or sustained muscle contractions (muscular dystonia) can be caused by a number of symptoms, including cerebral palsy, encephalitis, drug side effects, hepatic encephalopathy, and Huntington's chorea. Motor abnormalities can be reduced or disappeared during sleep, but are aggravated by emotional stress. Abnormal and sometimes odd postures may be manifestations of this exercise.

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a patient exhibiting dystonia.

Enlarged prostate

Botulinum toxin injections may be effective in men with benign prostatic hyperplasia. Thirty men with benign prostatic hyperplasia were involved in a randomized placebo-controlled trial. After baseline evaluation, each participant was injected 4 mL of solution into the prostate gland. Saline was administered to the control group and 200 U botulinum toxin A to the treated group. The results of each group were evaluated by comparing symptom scores, serum prostate-specific antibody concentrations, prostate volume, residual urine volume and peak urine flow.

Two months later, 13 patients in the treatment group and 3 in the control group noticed symptomatic relief (P = 0.007). In patients receiving botulinum toxin, the symptom score was reduced by 65% compared to the baseline value and the serum prostate-specific antigen concentration was reduced by 51% from the baseline value. In patients receiving saline, the symptom score and serum prostate-specific antigen concentrations were not significantly changed compared to baseline values and 1 month values. Follow-up was conducted for an average of 19.6 +/- 3.8 months (Maria et al , 2003, Urology 62: 259).

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a patient with symptoms of prostatic hyperplasia.

Hyperhidrosis

Hyperhidrosis is a medical condition in which you sweat excessively. A person with hyperhidrosis has a cool temperature and sweats even at rest. Sweating helps keep your body cool and is a perfectly natural phenomenon. People sweat more when the temperature is hot, when they exercise, or when they react to situations that are frustrating, angry, embarrassing, or scaring.

However, excessive sweating occurs without this induction. Hyperhidrosis has been shown to have overactive sweat glands. Uncontrollable sweating can lead to a very unpleasant condition, both physically and emotionally. When excessive sweating occurs in the hands, feet and armpits, it is called primary or local hyperhidrosis. Primary hyperhidrosis affects 2-3% of the population, but less than 40% of patients with this condition are consulted by a doctor. Most cases of primary hyperhidrosis have not been identified. This seems to be a family history. If sweating occurs due to another medical condition, it is called secondary hyperhidrosis. Sweating may occur in all parts of the body or may be confined to any one area. Symptoms causing secondary hyperhidrosis include acromegaly, hyperthyroidism, glucose control disorders, chromaffin cell carcinoma, carcinoid syndrome, cancer, tuberculosis, infection, menopause, spinal cord injury, stroke, Parkinson's disease, heart or lung disease, medication, Drug abuse or anxiety symptoms, but are not limited to these. The primary symptom of hyperhidrosis is wet soaking.

Botulinum toxin type A (BOTOX ^® ) was approved by the FDA in 2004 for the treatment of severe underarm arm sweating, a condition called primary axillary hyperhidrosis. Small amounts of purified botulinum toxin injected under the armpit temporarily block the nerves that stimulate sweating. Side effects are injection site pain and flu-like symptoms. BOTOX ^® , used for palm sweating, can cause temporary mild weakness and pain.

In one embodiment, the invention is directed to administering a botulinum nanoparticle composition, such as a microfluidized botulinum toxin nanoemulsion, to a patient with hyperhidrosis.

adduction

The following examples are intended only for the purpose of illustrating the specific embodiments targeted by the invention. The examples are not intended to be limiting in any way.

Example 1 Botulinum Nanoemulsion Formulation

This example illustrates the one embodiment of the nano-emulsion comprising a botulinum toxin (i.e., for example, BOTOX ^®) produced by the micro-fluidization.

Microfluidization formulations were prepared as follows:

1. Emulsify the mixture by mixing 5 g soybean oil and 5 g Tween 80 as needed (typically not required).

2. The mixture was stirred until it was added to 100 units of BOTOX ^® it is introduced into the human albumin matrix (Allergan, Irvine CA) deionized water / distilled water of 100 mL, and mixed uniformly.

3. The formulation of step 1 was added to the formulation of step 2 and stirred until uniformly mixed.

4. The formulation was homogenized for 1 minute (see particle distribution obtained in Table 1 and FIG. 1).

5. A single pass microfluidization procedure at 24,000 psi was performed using a Microfluidizer ^® treatment apparatus.

The particle diameter of the obtained nanoemulsion was evaluated using a Malvern Nano S particle sorter capable of classifying particles of about 0.6 to 6000 nm by size. The BOTOX ^® nanoemulsion formulations showed two particle diameter peaks with an average particle diameter of 95.33 nm (Table 2 and Figure 2).

Table 1 : Particle size distribution of homogenized BOTOX ^® microemulsion

Diameter (nm) burglar(%) Width (nm)  Z-Average: 3391 Peak 1 1512 100 76.6  PDI: 0.341 Peak 2 0 0 0  Intercept: 0.5852 Peak 3 0 0 0

Table 2: particle size distribution of the micro-fluidized BOTOX ^® microemulsion

Diameter (nm) burglar(%) Width (nm)  Z-Average: 95.33 Peak 1 134.2 76.49 31.03  PDI: 0.252 Peak 2 44.97 23.51 6.34  Intercept: 0.9659 Peak 3 0 0 0

Example 2: Infused BOTOX ^®  Muscle Relaxation Effect of Nanoemulsion

This example illustrates one embodiment of a BOTOX ^® nanoemulsion with potency comparable to free solution BOTOX ^® injection as saline.

The experimental design was compared with the following two BOTOX ^® formulations:

1) BOTOX ^® nanoemulsions prepared according to Example 1 were injected by intramuscular (IM) injection into the hind limbs (elbow muscles) of Swiss Webster female mice.

2) was injected BOTOX ^® saline as intramuscular (IM) injection into the hind leg muscle calf muscles of Swiss-Webster female mice.

Toe abduction score (DAS) analysis was used to determine local muscle weakening efficacy (Aoki, 1999). DAS values were assigned as follows: (0) flat foot, same toe spread as leg of control; (1) flat foot, toe abduction width is different compared to the foot of control, or two toes are in contact, and the other toes are fully extended; (2) a flat foot, with some space open at the end of all toes, or three toes touching; (3) when the feet are flat, the five toes are in contact; when the feet are bent, the four toes are in contact; (4) Bent feet, all five toes coming together.

BOTOX ^® to IM injection of the nano-emulsion and BOTOX ^® saline solution was evaluated 7 days DAS under simple blind protocol. DAS scores 1-2 were observed in both botulinum toxin nanoemulsions (3.96 U / 5 μl) and botulinum toxin saline (3.96 U / 5 μl). The control, nanoemulsion blank, had DAS (0). Each group (botulinum toxin nanoemulsion, saline and control) consisted of five (5) animals.

This information demonstrates that microfluidization technology does not destroy the functional properties of botulinum toxin and that botulinum toxin nanoemulsion is functionally effective, as evidenced by the injection of non-microfluidized botulinum toxin saline.

Example 3: transdermal BOTOX ^®  Muscle Relaxation Effect of Nanoemulsion

The examples demonstrate the therapeutic efficacy of botulinum nano-emulsion is applied percutaneously (i.e., for example, BOTOX ^® nano-emulsion).

BOTOX ^® nanoemulsions (9.9 U / 100 μl) prepared according to Example 1 were administered locally to the hind limb muscles of five (5) Swiss Webster female mice. Swiss Webster female mice of five (5), except that you omit the BOTOX ^® was administered the nano-emulsion equally prepared. For 7 days after treatment, a DAS score of 1 to 2, scored according to Example 2, was observed in the botulinum toxin nanoemulsion treatment group, but not in the control group. Skin deterioration (eg irritation, redness, etc.) was not observed at any point after treatment. The data show that botulinum toxin nanoemulsions are biologically active when administered transdermally in a manner similar to conventionally administered botulinum toxin injections.

Example 4: Muscle Relaxation Effect by Botulinum Nanoemulsion Administration: Controlled Comparison of Standard Injected Botulinum Versus Topical Botulinum Nanoemulsion in Mice

This example provides a controlled experiment to demonstrate that the application of the topical botulinum nanoemulsion of the invention can induce a muscle relaxation effect equivalent to a standard injected botulinum formulation (not a nanoemulsion).

Way

35 Swiss Webster mice, weighing about 20 g, were purchased from Charles River. Upon arrival, all animals were domesticated in their cages for one week (five mice per group were housed in a group as defined below), laid on the floor with standard cages and fed with Purina 5001 solid food. After 1 week, local muscle function was determined after application of the BOTOX ^® nanoemulsion prepared according to Example 1 using the Toe Abduction Score (DAS). In the DAS analysis, the animal's tail was lifted briefly (10 seconds) to induce a characteristic surprise response that stretched his hind limbs and shortened his hind toes. This analysis was conducted once a week for three weeks.

Three therapeutic formulations were prepared for the three treatment groups of mice: 1) BOTOX ^{® in} injection saline, 2) nanoemulsions containing BOTOX ^® and 3) all of the BOTOX ^® nanoemulsion except BOTOX ^® . BOTOX ^® and containing the nano-emulsion and also a process "the blank (blank)" nano-emulsion through a micro-fluidizer processing apparatus in the same way containing the following ingredients.

Therapeutic Paradigm

The BOTOX ^® were suspended in saline to 15 mice of group 1 (IM) with 10 U / 5 ㎕ per 1 kg body weight were treated by injection in the thigh muscle of the mouse hind limb muscles.

Group 2 by applying the 15 mice body weight 1 10 U / of 100 ㎕ BOTOX ^® per kg of the (topical) in the mouse skin in the upper hind limb muscle calf muscle was localized treatment.

By applying a blank containing no nano-emulsion BOTOX ^® to 15 mice in group 3 (control) in a mouse skin in the upper hind limb muscle calf muscle it was localized treatment.

evaluation

One week after injection and / or transdermal application, DAS analysis was used to determine the potential local muscle weakening effect of the treatment. This analysis was performed once a week for the next three weeks. After injection and / or transdermal application of BOTOX ^® or a control formulation, observers who did not participate in treatment scored the degree of change in toe failure on a 5-point scale (0 = normal to 4 = maximum reduction in toe extension and leg extension). .

Results and conclusion

After 1 week of treatment, the control mice treated with the blank nanoemulsion (group # 3) received 0.5 ± 0.3 on the Aoki scale, compared with the mice treated with the topical botulinum nanoemulsion formulation (group # 2). Obtained 2.8 ± 0.3 points on the Aoki scale (P <0.001). In contrast, mice injected with botulinum in saline (group # 1) obtained 3.5 ± 0.3 points. After 3 weeks of treatment, both groups of mice treated with topical botulinum nanoemulsion formulations and mice injected with botulinum in saline obtained control levels of Aoki scores expected from the public literature on injection botulinum. (This decrease in Aoki scale in mice has been repeatedly observed with botulinum, but when used in therapeutic doses in humans, anti-wrinkle effects have been seen for several months). In addition, skin exacerbations (eg irritation, redness, etc.) were not observed at any point after treatment.

In summary, these control data strongly suggest that topical botulinum nanoemulsion formulations deliver biological effects comparable to injectable botulinum.

Example 5 Administration of Botulinum Nanoparticle Compositions to Human Subjects for Wrinkle Relief

To prepare the topical botulinum nanoemulsion of the present invention and to determine whether the forehead muscle of the wrinkled forehead has a relieving effect (in much the same way as expected with administration of botulinum suspended in simple saline injected into the muscle), It was applied to the wrinkled human.

Way

Botulinum nanoemulsions were prepared using the following steps:

1. 800 mg soybean oil and 800 mg Tween 80 were stirred for 5 minutes in a sterile vial.

2. 8.4 mL of 0.9% saline was added with 4500 units of approved botulinum type A toxin agent. Stir for 20 minutes.

3. The sample was homogenized for 1 minute.

4. The sample was stirred for 20 minutes.

5. Microfluidized once to 23,000 psi.

Nanoemulsion was added to an equal volume of skin cream (base PCCA varnishing cream light) and vortexed into a uniform cream.

Due to over activity of the forehead muscles, patients with severe horizontal wrinkles were selected and treated. This patient had never been treated with botulinum products or dermal filler products. Prior to treatment, a licensed plastic surgeon assessed the patient using a 4-point wrinkle scale ("1" corresponds to "no wrinkles" and "4" corresponds to severe wrinkles). Patients were evaluated using this scale when the forehead attempts to contract forehead muscles to create maximum wrinkles by "resting" and lifting their eyebrows up as much as possible ("maximum eyebrow lifting").

The patient earned 4 points at rest and 4 points at the maximum lift of the eyebrows. The patient proved to have excellent movements to contract the forehead muscles. The patient was photographed with a digital SLR camera at rest and with the forehead lifted up and taken with digital video (FIG. 3A, maximum eyebrow lift before treatment).

 On the day of treatment, the patient was asked to wash his face with ivory soap and not use any facial makeup or sunscreen before coming to the doctor's office. In the clinic, a plastic surgeon applied a 0.6 CC nanoemulsion cream (as prepared in Example 1) to the forehead of the patient with forehead muscle distribution. The doctor applied the cream to the patient's forehead skin using a pipette and rubbed the skin with fingers (wearing surgical gloves) until the cream was no longer visible to the doctor's eyes. Patients were observed for three hours in the office. The patient was asked not to touch his forehead for 12 hours and then rinsed with ivory soap and water. Patients were followed up after 1 day and then at 1, 2, 4, 8 and 12 weeks. At follow-up visits, the physician assessed the patient's wrinkles at rest and with maximum brow lifting. Similarly, the doctor repeated the digital work, standardized in photographs and videos.

result

On the first week after treatment, the patient was unable to lift his eyebrows when asked to lift his eyebrows to the maximum and thus could not contract his forehead muscles (FIG. 3B). His wrinkle score was 2 points at rest and 2 points when he raised his eyebrows to the maximum. The physician clinically assessed that the treatment induced complete paralysis of the treated muscles, equivalent to the treatment performed by injecting botulinum toxin into another treatment area in another patient. The patient had slightly restored forehead movements at week 8, but continued to continue a significant decrease in his forehead movements at week 12 of observation.

The patient was able to move his facial muscles underneath the untreated skin area and the plastic surgeon could not observe side effects, including no skin changes immediately after treatment or at any follow-up visit. Similarly, the patient did not complain of adverse events including any skin changes (eg irritation, redness, etc.) at any point after treatment.

conclusion

In other words, the experiments showed significant biological and clinical effects comparable to those expected after standard treatment of botulinum (in simple saline) in which topical botulinum nanoemulsion formulations were injected into patients (compared by a plastic surgeon). Assessed] is strongly implied.

Example 6: Additional Botulinum Nanoparticle Composition Formulations

In some cases, various different botulinum nanoparticle compositions were prepared in the same manner as in Example 1, except that the equipment used, the applied pressure, the amount of botulinum added, and the volume of the prepared nanoparticle composition differed, which observed size variability. Can you explain why? The following average particle diameters and distributions were observed (Table 3):

Table 3 : Particle Size Distribution of Microfluidized BOTOX ^® Nanoemulsions

Example 7: Relationship between applied pressure and average particle diameter obtained

Premix formulations were prepared as in Example 1 (except for the absence of botulinum toxin), dispensed into 100 ml 4 aliquots A to D, and then passed through Microfluidizer ^® at different pressures, as shown in Table 4 below. As a result, different average particle diameters were obtained.

Table 4 : Particle diameters of BOTOX ^® nanoemulsions microfluidized at different pressures

Even range and area

Specific preferred embodiments of the invention have been described which are not limiting above. Those skilled in the art will recognize or appreciate that certain embodiments of the invention described herein may have various equivalent ranges without departing from routine experimentation. Those skilled in the art will recognize that various changes and modifications can be made in the above description without departing from the spirit or scope of the invention as defined in the following claims.

In the claims, the singular may also mean the plural concept unless otherwise stated or clear from the context. A range or description including “or” between one or more members of a group, unless stated otherwise or evident from the context, indicates that one, more, or all of the group members are present in, used in, or otherwise relevant to a given product or process. If it is considered satisfied. The invention includes embodiments in which exactly one group member is present, used or otherwise relevant to a given product or process. The invention also includes embodiments in which a plurality or all group members are present in, used in, or otherwise relevant to a given product or process. Also, it is to be understood that the present invention includes all modifications, combinations and substitutions from one or more claims or related parts of the description that incorporate one or more of the limitations, elements, clauses, technical terms, etc. into other claims. For example, any claim that depends on another claim may be modified to include one or more limitations on any other claim that depends on the same base claim. Also, where the claim refers to a composition, unless otherwise stated or unless it is apparent to those skilled in the art that contradictions or inconsistencies occur, a method of use of the composition for any purpose disclosed herein is included and is disclosed herein. It is to be understood that a method of making a composition according to any method of manufacture or other methods known in the art is included. The invention also includes compositions prepared according to the methods of making any of the compositions disclosed herein.

When components are listed, for example in Markish type, it is to be understood that each subgroup of components is also identified and any component (s) may be omitted from the group. It is also to be noted that the term "comprising" is intended to be open and may involve additional components or steps. In general, where the invention or aspects of the invention refer to particular components, features, steps, or the like, a particular embodiment of the invention or aspects of the invention may consist of or consist essentially of such components, features, steps, or the like. It will be understood that it is constructed. For purposes of simplicity, the embodiments have not been explicitly described herein as such. That is, for each embodiment of the invention that includes one or more components, features, steps, and the like, the invention also provides embodiments that consist or consist essentially of these components, features, steps, and the like.

If a range is given, the endpoint is included. Also, unless stated to the contrary, and apparent from the context, and / or understood by one of ordinary skill in the art, values expressed in ranges may be defined in any of the lower limits of the range unless otherwise specified. It should be understood that one tenth can be taken. Also, unless stated to the contrary, and apparent from the context, and / or understood by one of ordinary skill in the art, values expressed in ranges may take any subrange within the given range, with the endpoint of the subrange being ten minutes of the lower end of the range. It should be understood that it is expressed with the same accuracy as.

In addition, it should be understood that any specific embodiment of the invention may be expressly excluded from any one or more claims. Any embodiment, component, feature, application or aspect of the compositions and / or methods of the invention (such as any botulinum toxin, any oil, any surfactant, any dispersion medium, any nanoparticle, any Compositions comprising nanoparticles, any method of making nanoparticles, any route or location of administration, any purpose for administering the composition, etc.) may be excluded from any one or more claims. For the sake of brevity, not all embodiments are explicitly described herein except for one or more components, features, objects or aspects.

Claims (180)

As a nanoemulsion containing a particle group, The majority of the particles have a diameter of about 10 to about 300 nanometers, the nanoemulsion comprises one or more botulinum toxin. The nanoemulsion of claim 1, wherein the majority of particles have a diameter range of about 10 to about 250 nanometers. The nanoemulsion of claim 1, wherein the majority of particles range in diameter from about 10 to about 200 nanometers. The nanoemulsion of claim 1, wherein the majority of particles have a diameter range of about 10 to about 150 nanometers. The nanoemulsion of claim 1, wherein the majority of particles have a diameter range of about 10 to about 120 nanometers. The nanoemulsion of claim 1, wherein the majority of particles have a diameter range of about 10 to about 100 nanometers. The nanoemulsion of claim 1, wherein the majority of particles have a diameter range of about 10 to about 50 nanometers. The nanoemulsion of claim 1, wherein the particle population is substantially free of particles having a diameter greater than 300 nm. The nanoemulsion of claim 1, wherein less than 50% of the particles have a diameter of greater than 300 nm. The nanoemulsion of claim 1, wherein less than 25% of the particles have a diameter of greater than 300 nm. The nanoemulsion of claim 1, wherein less than 10% of the particles have a diameter of greater than 300 nm. The nanoemulsion of claim 1, wherein less than 5% of the particles have a diameter of greater than 300 nm. The nano-mersion of claim 1, wherein less than 1% of the particles have a diameter of greater than 300 nm. The nanoemulsion of claim 1, wherein the particle population is substantially free of particles having a diameter greater than 200 nm. The nanoemulsion of claim 1, wherein less than 50% of the particles have a diameter of greater than 200 nm. The nanoemulsion of claim 1, wherein less than 25% of the particles have a diameter of greater than 200 nm. The nanoemulsion of claim 1, wherein less than 10% of the particles have a diameter of greater than 200 nm. The nanoemulsion of claim 1, wherein less than 5% of the particles have a diameter of greater than 200 nm. The nanoemulsion of claim 1, wherein less than 1% of the particles have a diameter of greater than 200 nm. The nanoemulsion of claim 1, wherein the particle population is substantially free of particles having a diameter greater than 120 nm. The nanoemulsion of claim 1, wherein less than 50% of the particles have a diameter of greater than 120 nm. The nanoemulsion of claim 1, wherein less than 25% of the particles have a diameter of greater than 120 nm. The nanoemulsion of claim 1, wherein less than 10% of the particles have a diameter of greater than 120 nm. The nanoemulsion of claim 1, wherein less than 5% of the particles have a diameter of greater than 120 nm. The nanoemulsion of claim 1, wherein less than 1% of the particles have a diameter of greater than 120 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 600 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 500 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 400 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 300 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 200 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 100 nm. The nanoemulsion of claim 1, wherein the difference between the minimum and maximum particle diameters does not exceed about 50 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 300 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 200 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 150 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 100 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 75 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter of 50 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 100 to 300 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 50 to 250 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 60 to 200 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 65 to 150 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 70 to 130 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 80 to 110 nm. The nanoemulsion of claim 1, wherein the particles have an average diameter in the range of 90 to 100 nm. The nanoemulsion of claim 1, wherein the nanoemulsion is substantially free of toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion comprises less than 50% toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion comprises less than 25% toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion comprises less than 10% toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion comprises less than 5% toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion comprises less than 1% toxic solvent. The nanoemulsion of claim 1, wherein the nanoemulsion is stable. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least one day. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least two weeks. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least two months. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least 5 months. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least 12 months. The nanoemulsion of claim 1, wherein the majority of particles are stable for at least 24 months. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to high shear forces. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to high shear for less than 10 minutes. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by exposure to high shear for less than 2 minutes. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by exposure to high shear for less than one minute. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to high shear for less than 30 seconds. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by exposure to a pressure above 3,000 psi. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to a pressure greater than 10,000 psi. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to a pressure greater than 18,000 psi. The nanoemulsion of claim 1 wherein the nanoemulsion is produced by exposure to excess pressure of 24,000 psi. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by microfluidization. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by microfluidization at a pressure above 3,000 psi. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by microfluidization at a pressure above 10,000 psi. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by microfluidization at a pressure above 18,000 psi. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by microfluidization at a pressure above 24,000 psi. 73. The nanoemulsion of any of claims 68 to 72, wherein the microfluidization is a single-pass microfluidization. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by cavitation. The nanoemulsion of claim 1, wherein the nanoemulsion is produced by high pressure homogenization. The nanoemulsion of claim 1, wherein the botulinum toxin is encapsulated in the particle. The nanoemulsion of claim 1, wherein the botulinum toxin is adsorbed on the particle surface. The nanoemulsion of claim 1, wherein the botulinum toxin is associated with the particle interface. The nanoemulsion of claim 1, wherein the botulinum toxin is selected from the group comprising A, B, C ₁ , C ₂ , D, F and G forms. The nanoemulsion of claim 1 wherein the botulinum toxin is a type A botulinum toxin. The nanoemulsion of claim 1, wherein the botulinum toxin is a botulinum toxin complex. 82. The nanoemulsion of claim 81 wherein the botulinum toxin complex comprises non-toxic erythrocyte aggregate protein and non-toxic erythrocyte aggregate protein. The nanoemulsion of claim 1, wherein the botulinum toxin is incorporated into the albumin substrate. The nanoemulsion of claim 1, wherein the albumin is human albumin. The nanoemulsion of claim 1, wherein the botulinum toxin is a purified botulinum toxin protein or fragment thereof. The nanoemulsion of claim 1, wherein the botulinum toxin is isolated from or substantially separated from other proteins. The nanoemulsion of claim 1, wherein the botulinum toxin is isolated from or substantially separated from the non-toxin protein. The nanoemulsion of claim 1, wherein the botulinum toxin is isolated from Clostridium botulinum . The nanoemulsion of claim 1 wherein the botulinum toxin is chemically synthesized. The nanoemulsion of claim 1, wherein the botulinum toxin is recombinantly produced. The nanoemulsion of claim 1, wherein the nanoemulsion can penetrate the skin with or without altering the skin. The nanoemulsion of claim 1, wherein the nanoemulsion can penetrate the skin without the use of a skin penetration enhancer or abrasive. The nanoemulsion of claim 1, wherein the nanoemulsion can penetrate the upper layers of the skin without the use of skin penetration enhancers or abrasives. 94. The nanoemulsion of claim 93, wherein the upper layer of skin is the stratum corneum surface. 95. The nanoemulsion of claim 93, wherein the upper layer of skin comprises dermal pores. 95. The nanoemulsion of claim 93, wherein the upper layer of skin comprises the dermis. The nanoemulsion of claim 1, wherein the nanoemulsion can penetrate the skin without the use of chemical penetration enhancers or abrasives. The nanoemulsion of claim 1, wherein the nanoemulsion can penetrate the skin without the use of mechanical penetration enhancers or abrasives. The nanoemulsion of claim 1, wherein the particles can penetrate the skin with or without altering the skin. The nanoemulsion of claim 1, wherein the particles can penetrate the skin without the use of a skin penetration enhancer or abrasive. The nanoemulsion of claim 1, wherein the botulinum toxin can penetrate the skin with or without altering the skin. The nanoemulsion of claim 1, wherein the botulinum toxin can penetrate the skin without the use of a skin penetration enhancer or abrasive. The nanoemulsion of claim 1, wherein the nanoemulsion comprises an oil. 103. The oil of claim 103, wherein the oil is almond, apricot kernel, avocado, babasu palm, bergamot, black current seed, borage, cade, chamomile, canola, Caraway, Carnauba, Castor, Cinnamon, Cocoa Butter, Coconut, Cod Liver, Coffee, Corn, Cotton Seed, Emu, Eucalyptus, Evening Primrose, Fish, Flax Seed, Geraniol, Calabash (gourd), grape seeds, hazelnuts, hyssop, isopropyl myristate, jojoba, cucuit nuts, labandine, lavender, lemon, litsea cubeba, mecademia nut, Mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed , Rape seed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, Sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut and malt oil, and combinations thereof Nanoemulsion. 104. The nanoemulsion of claim 103, wherein the oil is soybean oil. 104. The oil of claim 103, wherein the oil is butyl stearate, caprylic acid triglycerides, capric acid triglycerides, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol , Nanoemulsions selected from the group consisting of silicone oils and combinations thereof. The nanoemulsion of claim 1, wherein the nanoemulsion comprises at least one oil. The nanoemulsion of claim 1, wherein the nanoemulsion comprises a surfactant. 109. The nanoemulsion of claim 108, wherein the surfactant is a nonionic detergent. 109. The method of claim 108, wherein the surfactant is phosphoglyceride; Phosphatidylcholine; Dipalmitoyl phosphatidylcholine (DPPC); Dioleylphosphatidyl ethanolamine (DOPE); Dioleyloxypropyltriethylammonium (DOTMA); Dioleoylphosphatidylcholine; cholesterol; Cholesterol esters; Diacylglycerols; Diacylglycerol succinate; Diphosphatidyl glycerol (DPPG); Hexanedecanol; Fatty alcohols such as polyethylene glycol (PEG); Polyoxyethylene-9-lauryl ether; Surfactant fatty acids such as palmitic acid or oleic acid; fatty acid; Fatty acid monoglycerides; Fatty acid diglycerides; Fatty acid amides; Sorbitan trioleate (Span 85) glycocholate; Sorbitan monolaurate (Span 20); Polysorbate 20 (Tween-20); Polysorbate 60 (Tween-60); Polysorbate 65 (Tween-65); Polysorbate 80 (Tween-80); Polysorbate 85 (Tween-85); Polyoxyethylene monostearate; Supactin; Poloxomers; Sorbitan fatty acid esters such as sorbitan trioleate; lecithin; Lysocithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebroside; Dicetylphosphate; Dipalmitoylphosphatidylglycerol; Stearylamine; Dodecylamine; Hexadecyl-amine; Acetyl palmitate; Glycerol ricinoleate; Hexadecyl stearate; Isopropyl myristate; Tyloxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipids; Synthetic and / or natural detergents with high surfactant properties; Deoxycholate; Cyclodextrins; Chaotropic salts; Ion pairing agents; And nanocombines thereof. 109. The nanoemulsion of claim 108, wherein the surfactant is Tween 80. The nanoemulsion of claim 1, wherein the nanoemulsion comprises at least one surfactant. The nanoemulsion of claim 1, wherein the nanoemulsion comprises an oil and a surfactant. 116. The nanoemulsion of claim 113, wherein the oil and the surfactant are present in a ratio ranging from 0.5 to 2.0. 116. The nanoemulsion of claim 113, wherein the oil and the surfactant are present in proportions ranging from 0.5 to 1.5. 116. The nanoemulsion of claim 113, wherein the oil and the surfactant are present in a ratio ranging from 0.5 to 1.0. 116. The nanoemulsion of claim 113, wherein the oil and the surfactant are present in a ratio ranging from 1.0 to 2.0. 116. The nanoemulsion of claim 113, wherein the oil and the surfactant are present in a ratio ranging from 1.5 to 2.0. 116. The nanoemulsion of claim 113, wherein the% of oil in the nanoemulsion is in the range of 1-30%. 116. The nanoemulsion of claim 113, wherein the% of oil in the nanoemulsion is in the range of 1-20%. 116. The nanoemulsion of claim 113, wherein the% of oil in the nanoemulsion is in the range of 1-10%. 116. The nanoemulsion of claim 113, wherein the% of oil in the nanoemulsion is about 8%. 116. The nanoemulsion of claim 113, wherein the% of oil in the nanoemulsion is about 5%. 116. The nanoemulsion of claim 113, wherein the percentage of surfactant in the nanoemulsion is in the range of 1-30%. 116. The nanoemulsion of claim 113, wherein the percentage of surfactant in the nanoemulsion is in the range of 1-20%. 116. The nanoemulsion of claim 113, wherein the percentage of surfactant in the nanoemulsion is in the range of 1-10%. 116. The nanoemulsion of claim 113, wherein the% of surfactant in the nanoemulsion is about 8%. 116. The nanoemulsion of claim 113, wherein the% of surfactant in the nanoemulsion is about 5%. 113. The oil of claim 113, wherein the oil is almond, apricot kernel, avocado, babassu palm, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, Cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, calabash, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, cucuit nut, ravandine, lavender, Lemon, Livia Kubeva, Mecademia Nut, Mallow, Mango Seed, Meadowfoam Seed, Mink, Nutmeg, Olive, Orange, Orange Rough, Palm, Palm Core, Peach Core, Peanut, Poppy Seed, Pumpkin Seed, Rape Seeds, rice bran, rosemary, safflower, sandalwood, sascuana, saber, sea buckthorn, sesame seeds, shea butter, silicone, soybeans, sunflower, tea tree, thistle, chubbaki, vetiver, walnut and malt oil, and combinations Selected from the group consisting of Nano-emulsions. 116. The nanoemulsion of claim 113, wherein the oil is soybean oil. 115. The oil of claim 113, wherein the oil is butyl stearate, caprylic acid triglycerides, capric acid triglycerides, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol , Nanoemulsions selected from the group consisting of silicone oils and combinations thereof. 116. The nanoemulsion of claim 113, wherein the surfactant is a nonionic detergent. 113. The method of claim 113, wherein the surfactant is selected from phosphoglycerides; Phosphatidylcholine; Dipalmitoyl phosphatidylcholine (DPPC); Dioleylphosphatidyl ethanolamine (DOPE); Dioleyloxypropyltriethylammonium (DOTMA); Dioleoylphosphatidylcholine; cholesterol; Cholesterol esters; Diacylglycerols; Diacylglycerol succinate; Diphosphatidyl glycerol (DPPG); Hexanedecanol; Fatty alcohols such as polyethylene glycol (PEG); Polyoxyethylene-9-lauryl ether; Surfactant fatty acids such as palmitic acid or oleic acid; fatty acid; Fatty acid monoglycerides; Fatty acid diglycerides; Fatty acid amides; Sorbitan trioleate (Span 85) glycocholate; Sorbitan monolaurate (Span 20); Polysorbate 20 (Tween-20); Polysorbate 60 (Tween-60); Polysorbate 65 (Tween-65); Polysorbate 80 (Tween-80); Polysorbate 85 (Tween-85); Polyoxyethylene monostearate; Supactin; Poloxomers; Sorbitan fatty acid esters such as sorbitan trioleate; lecithin; Lysocithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebroside; Dicetylphosphate; Dipalmitoylphosphatidylglycerol; Stearylamine; Dodecylamine; Hexadecyl-amine; Acetyl palmitate; Glycerol ricinoleate; Hexadecyl stearate; Isopropyl myristate; Tyloxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipids; Synthetic and / or natural detergents with high surfactant properties; Deoxycholate; Cyclodextrins; Chaotropic salts; Ion pairing agents; And nanocombines thereof. 113. The nanoemulsion of claim 113 wherein the surfactant is Tween 80. The nanoemulsion of claim 1, wherein the nanoemulsion comprises oily particles dispersed in an aqueous dispersion medium. 137. The aqueous dispersion medium of claim 135, wherein the aqueous dispersion medium is water, saline solution, phosphate buffered saline, short chain alcohol, 5% dextrose, Ringer's solution, Lactated Ringer's injection, Lactated Ringer + 5% dextrose Nanoemulsion selected from the group consisting of injection solution, acylation Ringer injection solution, Nomosol-M (Isolyte E) and combinations thereof. 137. The nanoemulsion of claim 135, wherein the aqueous dispersion medium is water. 138. The oily particles of claim 135, wherein the oily particles are almonds, apricot kernels, avocados, babasu palm, bergamot, blackcurrant seeds, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut , Cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flax seed, geraniol, calabash, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, cucunut, labanine, lavender , Lemon, livia cubeva, mechaemia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange rupee, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, Selected from the group consisting of rapeseed, rice bran, rosemary, safflower, sandalwood, sascuana, saber, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, chubaki, vetiver, walnut or malt oil Containing oil Nano-emulsions. 136. The nanoemulsion of claim 135, wherein the oily particles comprise soybean oil. 137. The oily particle of claim 135, wherein the oily particles are butyl stearate, caprylic acid triglycerides, capric acid triglycerides, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl A nanoemulsion comprising an oil selected from the group consisting of alcohols, silicone oils and combinations thereof. The nanoemulsion of claim 1, wherein the nanoemulsion comprises aqueous particles dispersed in an oily dispersion medium. 142. The aqueous particle of claim 141, wherein the aqueous particles are water, saline, phosphate buffered saline, short chain alcohol, 5% dextrose, Ringer's solution, lactic acid-added Ringer's injection, lactic acid-added Ringer + 5% dextrose injection, acylated Ringer's injection, nomo Nanoemulsion comprising an aqueous material selected from the group consisting of Solmo-M (Normosol-M) and Isolete E (Isolyte E). 143. The nanoemulsion of claim 141 wherein the aqueous particles comprise water. 141. The oily dispersion medium according to claim 141, wherein the oily dispersion medium is almond, apricot kernel, avocado, babasu palm, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, Coconut, Cod Liver, Coffee, Corn, Cotton Seed, Emu, Eucalyptus, Evening Primrose, Fish, Flax Seed, Geraniol, Calabash, Grape Seed, Hazelnut, Hyssop, Isopropyl Myristate, Jojoba, Cucuit Nut, Labandine, Lavender, lemon, Licia cubeva, meca demia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange rupee, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed Rapeseed, rice bran, rosemary, safflower, sandalwood, sascuana, sabery, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, chubaki, vetiver, walnut and malt and their Line from the group consisting of blends A nano-emulsion to be. 143. The nanoemulsion of claim 141, wherein the oily dispersion medium is soybean oil. 141. The oil of claim 141, wherein the oil is butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol , Nanoemulsions selected from the group consisting of silicone oils and combinations thereof. A nanoemulsion comprising a population of particles having a diameter of about 10 to about 300 nanometers, wherein the nanoemulsion comprises one or more botulinum toxins, the botulinum toxins of the complex comprising a botulinum toxin associated with a non-toxin protein. Nanoemulsion which is part. A nanoemulsion comprising a population of particles having a diameter of about 10 to about 300 nanometers, wherein the nanoemulsion is produced by exposure to high shear forces, the nanoemulsion comprising one or more botulinum toxins, one or more oils, and one or more nonionic detergents. Wherein the botulinum toxin is incorporated into the human albumin matrix. As a nanoemulsion, A particle group having a diameter of about 10 to about 300 nanometers; One or more botulinum toxins; One or more oils; And 1 or less types of surfactant Including, The nanoemulsion is a nanoemulsion produced by exposure to high shear force. 42. A pharmaceutical composition comprising the nanoemulsion of any one of claims 1, 21, 31 or 41. 151. The composition of claim 150, wherein the composition is selected from the group consisting of creams, lotions, gels, ointments, sprays, powders, emollients, and combinations thereof. 151. The composition of claim 150, wherein the composition is a cream. A method of transdermally administering a botulinum toxin to a subject, (a) providing a composition comprising a botulinum toxin; And (b) administering the composition to the skin of the subject How to include. 153. The method of claim 153, wherein the composition is administered transdermally using an adhesive patch. 153. The method of claim 153, wherein the composition is administered transdermally using a spatula. 153. The method of claim 153, wherein the composition is administered transdermally using a swab. 153. The method of claim 153, wherein the composition is administered transdermally using a needleless syringe. 153. The method of claim 153, wherein the composition is administered transdermally using gloved fingers. 153. The method of claim 153, wherein the composition is administered transdermally using an unprotected finger. 153. The method of claim 153, wherein the composition is administered transdermally using a device that allows applying the composition to a target site on the skin without applying the composition to a non-target site on the skin. 153. The method of claim 153, wherein all of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 99% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 95% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 90% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 75% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 50% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 25% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 10% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein at least 1% of the botulinum toxin penetrates the skin. 153. The method of claim 153, wherein the composition is administered to treat wrinkles, facial lines and / or neck lines. 172. The wrinkle, facial fold, and / or neck folds of claim 170 wherein the hyperkinetic facial folds, facial folds, broad neck strips, decollete strips

And combinations thereof. 171. The method of claim 171, wherein the facial wrinkles comprise at least one of a forehead area, a glans area, a frontal area, a temporal area, a skin rhytid area, and a periorbital area. 153. The method of claim 153, wherein the composition is administered to treat hyperhidrosis. A method of transdermally administering a botulinum toxin to a subject, (a) (i) subject; (ii) a composition comprising the nanoemulsion of any one of claims 1, 21, 31 or 41. Providing a; And (b) administering the composition to the skin of the subject How to include. A method of treating wrinkles, facial lines and / or neck lines, (a) subjects exhibiting symptoms of (i) wrinkles, facial lines and / or neck lines; (ii) a composition comprising the nanoemulsion of any one of claims 1, 21, 31 or 41. Providing a; And (b) administering the composition to the subject's skin to reduce symptoms How to include. A method of delaying the occurrence of wrinkles, facial wrinkles and / or neck wrinkles, (a) (i) subjects exhibiting no symptoms of wrinkles, facial wrinkles and / or neck wrinkles; (ii) a composition comprising the nanoemulsion of any one of claims 1, 21, 31 or 41. Providing a; And (b) administering the composition to the subject's skin to delay the onset of symptoms How to include. As a method of preparing a nanoemulsion, (a) (i) oils; (ii) surfactants; And (iii) aqueous phase Providing a premix comprising a; And (b) subjecting the premix to high shear or high pressure homogenization for a period of time under conditions to achieve the nanoparticle composition of any one of claims 1, 21, 31, or 41. How to include. 178. The method of claim 177, wherein the premix comprises botulinum toxin. As a method of preparing a namo emulsion, (a) (i) oils; (ii) surfactants; And (iii) aqueous phase Providing a premix comprising a; (b) forming a solution comprising the premix; And (c) subjecting the premix to high shear or high pressure homogenization for a period of time under conditions to achieve the nanoparticle composition of any one of claims 1, 21, 31, or 41. How to include. As a method of preparing a nanoemulsion, (a) (iv) oils; (v) surfactants; (vi) an aqueous phase; And (vii) botulinum toxin Providing a premix comprising a; (b) forming a solution comprising the premix; And (c) subjecting the solution to high shear or high pressure homogenization for a period of time under conditions to achieve the nanoparticle composition of any one of claims 1, 21, 31, or 41. Wherein the oil and surfactant are present in a ratio ranging from 0.5 to 2.0 (weight); High shear forces are produced by microfluidization.

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